Using the MTT viability assay, the dose-dependent effects of Probenecid were tested on HepG2 cells, treated for 24 and 48 h. Probenecid did not exhibit significant cellular toxicity up to 250µM for 48 h (**Figure 1A**). Probenecid-treated HepG2 cells did not show morphological alterations (in terms of the number of vacuoles and shape) compared to untreated HepG2 cells (**Figure 1B**).

In **Figures 2A,B** the effect of the ABCC6 inhibition on CD73 expression is shown. CD73 protein level was reduced of about 30% after treatment of cells with 250µM Probenecid for 48 h. Furthermore, CD73 expression increased of 30 and 40% when cells were treated with 10 and 100µM adenosine, respectively, and did not change even in the presence of Probenecid. As ATP is the substrate carried by the ABCC6, cells were incubated with ATP in the presence or absence of Probenecid. As previously seen on cells treated with adenosine, the expression of CD73 was increased in a dose-dependent manner (20 and 60% with ATP at 50 and 500µM, respectively), and once again did not change in the presence of Probenecid (**Figures 3A,B**).

In order to verify whether Probenecid was able to affect the transcript levels of some ABC transporters genes, a Real Time-PCR experiment was carried out (**Figure 4A**). No variation was observed for all the ABCs examined except for ABCC6, which was significantly reduced (about 30%) in HepG2 cells treated with

and ABCB1 in three independent experiments (means ± SD). \*\*\**p* < 0.001, Student's *t*-test. β-actin was used as a loading control. All the experiments were performed treating HepG2 cells with 250µM of Probenecid for 48 h. DMSO-treated cells were used as control.

FIGURE 5 | The effects of adenosine on ABCC6 protein expression, with or without Probenecid. (A,B) Densitometric analysis and representative western blot of the ABCC6 protein in three independent experiments (means ± SD). \*\**p* < 0.01, \*\*\**p* < 0.001, Student's *t*-test. β-actin was used as a loading control. All the experiments were performed treating HepG2 cells with adenosine and/or Probenecid for 48 h. DMSO-treated cells were used as control.

Probenecid. This result was also confirmed at protein level by western blotting analysis (**Figures 4B,C**).

**Figure 5** shows the effect of adenosine on the expression of ABCC6. The addition of different concentration of adenosine (10 and 100µM) increased the ABCC6 protein level about of 40 and 60%, respectively. In HepG2 cells treated only with Probenecid, the ABCC6 expression decreased by about 30%, but no changes were observed if Probenecid is used in association with adenosine.

### DISCUSSION

PXE is considered a metabolic disease caused by lack of circulating factors able to prevent mineralization. Jansen et al. (2013, 2014) proposed that this factor is the pyrophosphate derived from the ATP released by ABCC6. Although this hypothesis has been widely demonstrated and pyrophosphate is used to prevent soft tissue mineralization in PXE patients, little is known about the role played by extracellular AMP and by adenosine.

Previous studies in our laboratory showed that knockdown of ABCC6 leads to dysregulation of some genes including NT5E, which has been found under-expressed (Miglionico et al., 2014). In order to verify whether this finding was due to the lack of activation of purinergic pathway, in this study we evaluated if CD73 expression was regulated by the extracellular ATP and adenosine.

We demonstrated that Probenecid, an ABC transporters inhibitor, downregulated the expression of CD73, and adenosine increased its expression. Similar results were obtained by adding ATP, an ABCC6 substrate, which ultimately restores the levels of adenosine. Taken together, these results suggested that ABCC6, promoting the efflux of ATP from the cells, supplies the extracellular compartment with both the pyrophosphate, necessary to prevent the calcification, and purine nucleotides and adenosine, crucial mediators of numerous patho-physiological mechanisms (Burnstock, 2012, 2017).

The decrease of ABCC6 and CD73 expression by Probenecid and their increase by adenosine suggested a

### REFERENCES

role for ABCC6 in the regulation of the purinergic system and could explain the overlapping clinical symptoms of ACDC and PXE.

In conclusion, in this study we demonstrated that ABCC6 supplies the extracellular space of nucleosides and nucleotides and then could contribute to the purinergic pathway activation. In PXE patients only the effects due to lack of PPi are observed (Uitto et al., 2011) as the amount of adenosine to activate purinergic receptors is low compared to that of pyrophosphate to prevent mineralization (Sachdeva and Gupta, 2013; Orris et al., 2016). However, we cannot exclude that the contribution of the ABCC6 in the regulation of the purinergic system can become more important in some liver diseases.

### AUTHOR CONTRIBUTIONS

FM carried out the experiments, FC prepared stably expressed HEK293 cells, MP performed western blot analysis, MFA and RM analyzed the data and performed statistical analysis, FB and AO designed experiments and wrote the paper. All authors read and approved the final manuscript.

### ACKNOWLEDGMENTS

This work was supported by RIL funds provided by the University of Basilicata.

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmolb. 2018.00075/full#supplementary-material

Supplemental Figure S1 | Inhibition of efflux of doxorubicin by Probenecid. Doxorubicin efflux assay in HEK293 cells stably transfected with *ABCC6*-Flag-pcDNA in absence (•) or in presence (◦) of 1 mM Probenecid and in HEK293 cells transfected with Flag-pcDNA in absence (N) or in presence (1) of 1 mM Probenecid. Data are expressed as mean ± SD of three independent experiments. Significant differences at 30 min were determined by Student's *t*-test: ∗*p*-value < 0.05, ∗∗*p* < 0.01.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Martinelli, Cuviello, Pace, Armentano, Miglionico, Ostuni and Bisaccia. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# ENaC in Cholinergic Brush Cells

Chrissy Kandel 1 , Patricia Schmidt 1 , Alexander Perniss 1 , Maryam Keshavarz 1 , Paul Scholz 2 , Sabrina Osterloh2 , Mike Althaus 3 , Wolfgang Kummer 1 and Klaus Deckmann1 \*

1 Institute for Anatomy and Cell Biology, Justus-Liebig-University Giessen, Giessen, Germany, 2 Department of Cell Physiology, Ruhr-University Bochum, Bochum, Germany, 3 School of Natural and Environmental Sciences, Newcastle University, Newcastle upon Tyne, United Kingdom

Cholinergic polymodal chemosensory cells in the mammalian urethra (urethral brush cells = UBC) functionally express the canonical bitter and umami taste transduction signaling cascade. Here, we aimed to determine whether UBC are functionally equipped for the perception of salt through ENaC (epithelial sodium channel). Cholinergic UBC were isolated from ChAT-eGFP reporter mice (ChAT = choline acetyltransferase). RT-PCR showed mRNA expression of ENaC subunits Scnn1a, Scnn1b, and Scnn1g in urethral epithelium and isolated UBC. Scnn1a could also be detected by next generation sequencing in 4/6 (66%) single UBC, two of them also expressed the bitter receptor Tas2R108. Strong expression of Scnn1a was seen in some urothelial umbrella cells and in 65% of UBC (30/46 cells) in a Scnn1a reporter mouse strain. Intracellular [Ca2+] was recorded in isolated UBC stimulated with the bitter substance denatonium benzoate (25 mM), ATP (0.5 mM) and NaCl (50 mM, on top of 145 mM Na+ and 153 mM Cl− baseline in buffer); mannitol (150 mM) served as osmolarity control. NaCl, but not mannitol, evoked an increase in intracellular [Ca2+] in 70% of the tested UBC. The NaCl-induced effect was blocked by the ENaC inhibitor amiloride (IC50 = 0.47µM). When responses to both NaCl and denatonium were tested, all three possible positive response patterns occurred in a balanced distribution: 42% NaCl only, 33% denatonium only, 25% to both stimuli. A similar reaction pattern was observed with ATP and NaCl as test stimuli. About 22% of the UBC reacted to all three stimuli. Thus, NaCl evokes calcium responses in several UBC, likely involving an amiloride-sensitive channel containing α-ENaC. This feature does not define a new subpopulation of UBC, but rather emphasizes their polymodal character. The actual function of α-ENaC in cholinergic UBC—salt perception, homeostatic ion transport, mechanoreception—remains to be determined.

Keywords: chemosensory cells, cholinergic, ENaC, urethra, urethral brush cells, salt

## INTRODUCTION

Bitter, sweet, umami, salty, sour, and fatty are the six recognized taste qualities detected by taste buds (Chaudhari and Roper, 2010). In type II sensory cells in the oropharyngeal taste buds, bitter, sweet, and umami perception is mediated by the canonical taste transduction signaling cascade, including G protein-coupled taste receptors, the taste-specific G protein α-gustducin, phospholipase Cβ2 (PLCβ2), and the transient potential receptor cation channel subfamily M member 5 (TRPM5) (Chaudhari and Roper, 2010). Other classes of G proteincoupled receptors respond to short- and long-chain fatty acids (Chaudhari and Roper, 2010).

#### Edited by:

Cesare Indiveri, University of Calabria, Italy

#### Reviewed by:

Vito De Pinto, Università degli Studi di Catania, Italy Diego Alvarez de la Rosa, Universidad de La Laguna, Spain

\*Correspondence:

Klaus Deckmann klaus.deckmann@ anatomie.med.uni-giessen.de

#### Specialty section:

This article was submitted to Cellular Biochemistry, a section of the journal Frontiers in Cell and Developmental Biology

> Received: 29 March 2018 Accepted: 25 July 2018 Published: 15 August 2018

#### Citation:

Kandel C, Schmidt P, Perniss A, Keshavarz M, Scholz P, Osterloh S, Althaus M, Kummer W and Deckmann K (2018) ENaC in Cholinergic Brush Cells. Front. Cell Dev. Biol. 6:89. doi: 10.3389/fcell.2018.00089 In contrast, acid (protons) and salt (sodium chloride) are monitored by ion channels, directly leading to depolarization of the taste cell. Nonselective cation channels formed by polycystic kidney disease 2-like 1 protein (PKD2L1) and polycystic kidney disease 2-like 3 protein (PKD1L3) were proposed as candidates for sour taste receptors (Huang et al., 2006; Ishimaru et al., 2006; Lopezjimenez et al., 2006; Chaudhari and Roper, 2010). An ion channel that is long been thought to mediate salt perception is the amiloride-sensitive epithelial sodium channel, ENaC (Heck et al., 1984; Avenet and Lindemann, 1988; Lindemann et al., 1998; Lin et al., 1999; Lindemann, 2001; Chandrashekar et al., 2010). It is predominantly expressed in epithelial cells of the colon, lung, kidney, sweat and salivary glands, where it is a major regulator of sodium absorption and, thereby, essential for fluid homeostasis (Duc et al., 1994; McDonald et al., 1995; Garty and Palmer, 1997). ENaC is also expressed in the urothelium (Carattino et al., 2005; Du et al., 2007; Birder et al., 2010; Birder and Andersson, 2013). The canonical heteromeric ion channel consists of three subunits (α, β, γ) (Canessa et al., 1994b), encoded by the genes Scnn1a, Scnn1b, and Scnn1c. A fourth δ-subunit with distinct characteristics was identified and the presence of this subunit changes the biophysical characteristics as well as molecular regulation of this ion channel. Mice, however, lack a functional gene for this subunit and its physiological function remains unclear (Giraldez et al., 2012; Wichmann et al., 2018). ENaC is a constitutively active ion channel. Still, its expression, membrane abundance and open probability are tightly regulated by extrinsic and intrinsic factors. These include hormones, intracellular kinases and intramembrane lipids, as well as the extracellular sodium concentration, pH and mechanical stimuli (Chraïbi and Horisberger, 2002; Althaus et al., 2007; Baines, 2013; Kleyman et al., 2018). The ion conductivity of αβγ-ENaC is limited to monovalent cations (Li+ > Na+ > K +) (Kellenberger and Schild, 2002).

Extraoral chemosensory cells, monitoring the composition of the mucosal lining fluid, have been described in the respiratory, gastrointestinal and urogenital tract. Like type II taste cells, they express the canonical taste transduction signaling cascade (taste receptors, α-gustducin, PLCβ2, TRPM5) (Höfer et al., 1996; Höfer and Drenckhahn, 1998; Finger et al., 2003; Krasteva et al., 2011, 2012; Deckmann et al., 2014; Schütz et al., 2015). They respond to bitter substances and bacterial products with a release of acetylcholine and initiate avoidance reflexes, thereby apparently serving as sentinels situated at entrances into the body (Finger and Kinnamon, 2011; Lee and Cohen, 2015; Deckmann and Kummer, 2016). These cholinergic epithelial cells also express villin, a structural protein of microvilli. Such cells have originally been termed "brush cells" in the respiratory tract, and this term has also been adopted to the villin-positive, cholinergic chemosensory cells of the urethra (urethral brush cells = UBC) (Deckmann et al., 2014, 2015). In line with the sentinel concept, UBC respond to heat-inactivated uropathogenic Escherichia coli and are connected to sensory nerve fibers (Deckmann et al., 2014). Bitter application into the urethral lumen reflexively triggers enhanced detrusor activity, which has been interpreted as a protective reflex, as potential hazardous content is expelled from the urethra through micturition (Deckmann et al., 2014; Kummer and Deckmann, 2017).

Most cholinergic UBC are polymodal chemosensory cells, responding both to bitter substances and to glutamate with an increase in intracellular calcium concentration ([Ca2+]i) (Deckmann et al., 2014). This discriminates them from type II taste bud cells, which are generally responsive either to bitter, representing an aversive stimulus, or to umami, an attractive stimulus (Nelson et al., 2001; Chaudhari and Roper, 2010). At the urethral mucosa, both stimuli represent a potential danger signal, since many bacterial products have bitter quality and glutamate (umami) facilitates bacterial growth in urine. Here, we aimed to determine whether their polymodal properties extend beyond taste receptor mediated qualities, focusing upon the perception of salt.

### MATERIALS AND METHODS

### Animals

Mice expressing enhanced green fluorescent protein (eGFP) under the control of the promoter of the acetylcholine synthesizing enzyme, choline acetyltransferase, (ChAT-eGFP; B6.Cg-Tg(RP23-268L19-EGFP)2Mik/J; Stock No. 007902) were obtained from Jackson Laboratory (Bar Habor, ME, USA). Mice expressing tdTomato, a bright red fluorescent protein, under the control of the promotor of Scnn1a, the coding gene sequence of α-ENaC (Scnn1a/tdTomato; Guy et al., 2015) were kindly provided by J. Guy and J. Staiger (Institute for Neuroanatomy, University Medical Center Goettingen, Georg-August-University Goettingen, Germany). This study was carried out in accordance with the recommendations of European Communities Council Directive of 24th November 1986 (86/609/EEC). The protocol was approved by the local authorities (Animal Welfare Officer at the University of Giessen and the Committee for Animal Welfare, Dept. V54, Regierungspräsidium Giessen, Germany; reference no. 572\_M).

### Cell Isolation

Cell isolation was performed as described previously (Deckmann et al., 2014). In brief: Urethrae were dissected, cut into small pieces, and enzymatically digested in dispase (2 mg/mL; Sigma-Aldrich/Merck, Darmstadt, Germany) and trypsin/PBS (1:1, Invitrogen, Carlsbad, CA, USA). After mechanical dissociation, cells were separated through a cell strainer (pore size 70µm; BD Bioscience, Franklin Lakes, NJ, USA). The ChAT promotor is constitutively active in cholinergic chemosensory cells (Tallini et al., 2006). Hence, UBC constitutively express eGFP which served to sort them via FACS and to identify them with a fluorescence microscope.

### RT-PCR

Total RNA from dissected urethra or pooled isolated cells (n = 4 samples, sorting based on ChAT-eGFP expression by FACS; BD

**Abbreviations:** AU, arbitrary units; ChAT, choline acetyltransferase; Den, denatonium benzoate; eGFP, enhanced green fluorescent protein; ENaC, epithelial sodium channel; [Ca2+]I , intracellular calcium concentration; TRPM5, transient receptor potential cation channel subfamily M member 5; UBC, urethral brush cell.

FASCARIA III cell sorter, settings and analysis were performed with a BD FACSDiva v6.1.3; BD Bioscience, Franklin Lakes, NJ, USA) was extracted using the Qiagen RNeasy Micro Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. Extracted total RNA from kidney was used as positive control. RT-PCR was performed as described previously (primer sequences: **Table S1**; Deckmann et al., 2014).

### Next Generation Sequencing

Next generation sequencing was performed as described elsewhere (Scholz et al., 2016). In brief: isolated single eGFPpositive cells were identified, picked and transferred to a PCR tube using a combined confocal laser-scanning/patch-clamp setup (Leica TCS SP5, Leica Microsystems/Luigs-Neumann, Wetzlar/Ratingen, Germany). Cell lysis, cDNA generation and amplification were performed using the Sigma SeqPlex RNA Amplification Kit (Sigma-Aldrich/Merck, Darmstadt, Germany). For library preparation, the Illumina Nextera XT DNA sample preparation protocol (Part # 15031942 Rev. C) was used. Samples run together with a 2 × 75 bp read length using the MiSeq Reagent Kit v3 (150 cycles) and the Illumina MiSeq Desktop Sequencer (Illumina, San Diego, CA, USA). The sequencing reads were aligned to the mm9 reference genome and transcriptome using TopHat2 (2.0.9). The TopHat output files were saved in BAM format and evaluated by Cuffdiff2 (2.1.1). All samples were compared and evaluated in one calculation cycle, allowing the algorithm to estimate the Fragments Per Kilobase Million (FPKM) values at the transcript level resolution and to control for variability across the replicate libraries.

### Immunohistochemistry and Whole-Mount Immunostaining

Specimen preparations and analyses were performed as described previously (Krasteva et al., 2011). In brief: urethrae used for immunohistochemistry (N = 3) and gall bladders used for whole-mount immunostaining (N = 2) were fixed using transcardiac perfusion with Zamboni solution (2% paraformaldehyde/15% saturated picric acid in 0.1 M phosphate buffer, pH 7.4). Fixed organs were dissected, washed in 0.1 M phosphate buffer (0.1 M NaH2PO4; 0.1 M Na2HPO4), and either incubated overnight in 18% sucrose in 0.1 M phosphate buffer and frozen in liquid nitrogen or mounted on a block of silicon elastomer using insect pins. Primary antibody was applied to 4–18 tissue sections from every individual animal. Primary antibodies were chicken anti-RFP (NBP1- 97371, 1:200 dilution; Novus Biologicals, Littleton, CO, USA) and rabbit anti-TPRM5 (1:2,000) (Kaske et al., 2007). Secondary antibodies were goat-anti rabbit Ig conjugated to Alexa 488 (1:500; Thermo Fisher Scientific Inc. Waltham, MA, USA) and donkey-anti chicken Ig conjugated to Cy3 (1:2,000; Dianova, Hamburg, Germany). Nuclei were labeled with 4′ ,6-diamidino-2-phenylindol (DAPI; 1µg/ml; Sigma-Aldrich/Merck, Darmstadt, Germany). All sections were rinsed and coverslipped with carbonate-buffered glycerol (pH 8.6). Sections were evaluated by epifluorescence microscopy (Axioplan 2, Zeiss, Wetzlar, Germany) or with a confocal laser scanning microscope (LSM 710, Zeiss, Wetzlar, Germany). Specificity of secondary reagents was validated by omission of primary antibodies.

### Measurement of Intracellular Calcium Concentration

Measurement of intracellular calcium concentration ([Ca2+]i) was performed as described previously (Deckmann et al., 2014). In brief: Isolated cells were loaded with the fluorescent calcium indicator Calcium Orange R AM (0.01 µg/µL; Thermo Fisher Scientific Inc., Waltham, MA, USA) and plated on coverslips. [Ca2+]i was analyzed with a confocal laser scanning microscope (LSM 710 with ZEN 2010 B SP1, Zeiss, Wetzlar, Germany). Lasers and filters were: eGFP: excitation with Argon laser at 488 nm; recording of emission at 495–553 nm with optical filters MBS-488/561/633; Calcium Orange: excitation with DPSS561-10 laser at 561 nm; recording of emission at 566-683 nm with optical filters MBS-558/561. Regions of interest were selected manually and fluorescence intensities at the start of the recording period were set arbitrarily at 100%. Test stimuli and concentrations were denatonium benzoate (25 mM; Molekula, Munich, Germany), ATP (0.5 mM; Sigma-Aldrich/Merck, Darmstadt, Germany) and NaCl (1–150 mM; Carl Roth, Karlsruhe, Germany), and inhibitors and controls included the osmolarity control mannitol (1–150 mM; Sigma-Aldrich/Merck, Darmstadt, Germany) and the ENaC inhibitor amiloride (0.01–100µM; Sigma-Aldrich/Merck, Darmstadt, Germany). All recordings were done during continuous superfusion with Tyrode III buffer (NaCl 130 mM; HEPES 10 mM; glucose 10 mM; KCl 5 mM; MgCl2 1 mM; CaCl2 8 mM; sodium pyruvate 10 mM; NaHCO3 5 mM; 2.5 mL/min; 37◦C). Stimuli were added under continuous flow of Tyrode III into the chamber, so that indicated concentrations were reached initially and then washed out. Since baseline concentration of Na+ in the buffer was 145 mM, the total concentration after addition of 1–150 mM ranged from 146 to 295 mM.

### Statistical Analysis

Data were analyzed for normal distribution by the Kolmogorov-Smirnov test. Multiple comparison analysis was performed by Kruskal-Wallis test followed by Dunn's Multiple Comparison Test. P ≤ 0.05 were regarded as statistically significant. Analyses were performed by GraphPad Prism 5 (GraphPad Software Inc., La Jolla, CA, USA).

### RESULTS AND DISCUSSION

RT-PCR revealed mRNA expression of the ENaC subunits Scnn1a, Scnn1b, and Scnn1g in the urethral epithelium (**Figure 1A**) and in pooled isolated UBC (**Figure 1B**). Next generation sequencing (NGS) of six isolated single eGFPpositive cells showed a heterogeneous expression pattern of Scnn1a, b, g (**Figure 2**). Scnn1a was detected in 4/6 cells (66.6%), Scnn1b and Scnn1g only in 1/6 cells. Canonical ENaC is composed of the α-, β-, and γ-subunit (Canessa et al., 1994a), but the ENaC α-subunit alone is able to

form amiloride-sensitive homomers in vitro (Canessa et al., 1994b).

To further validate Scnn1a expression in cholinergic UBC, urethral tissue sections of a Scnn1a reporter mouse strain were labeled for cholinergic UBC. In view of often experienced methodological problems in detecting ChAT by immunohistochemistry in peripheral cells, we set out to establish a technically more reliable marker for immunohistochemical detection of cholinergic UBC. Villinantibodies, an often used marker for brush cells in general, appeared not suitable for this purpose as there is a considerable number of villin-positive but ChAT- and TRPM5-negative slender epithelial cells in the murine urethra, in addition to the villin/ChAT/TRPM5-positive cells (Deckmann et al., 2014). These two phenotypes represent truly different cell populations, since genetic ablation of the transcription factor Skn-1a/Pou2f3 selectively prevents the development of TRPM5-positive (i.e., cholinergic UBC) but not of villinpositive but TRPM5-negative urethral cells (Yamashita et al., 2017).

We used TRPM5-immunolabeling as a marker for cholinergic UBC in Scnn1a-tdTomato reporter mice. In these mice, strong expression of Scnn1a was observed in several cells of the urethral epithelium (**Figures 3A,B**). Among them were umbrella cells, which build up the luminal lining in the proximal parts of the urethra being covered with an urothelium and which can be readily identified by virtue of their position and morphology. This is in line with the previously reported ENaCα-immunoreactivity at the luminal membrane of umbrella cells in the rat urinary bladder (Smith et al., 1998) and functional investigation of this cell type (McCloskey et al., 2017). Notably, this cell layer did not consistently express tdTomato with positive and negative umbrella cells occurring in a mosaic pattern (**Figure 3A**). Although heterogeneity of umbrella cells with respect to other characteristics such as uroplakin expression has also been reported in select localizations such as the human ureter (Riedel et al., 2005), this labeling pattern might reflect incomplete expression of tdTomato in potentially Scnn1a-expressing cells. To test for this possibility, we looked for tdTomato expression in the gall bladder whose mucosal surface is known for homogeneous ENaCα expression (Li et al., 2016). In two gall bladder whole-mount preparations, strong tdTomato expression was observed in epithelial cells covering only about 21% (case 1: 26.8%, case 2: 16.0%) of the mucosal surface whereas nearly 80% remained unlabeled (**Figures 3C,D**). Gall bladder whole-mounts were also incubated with TRPM5 antibody in order to label cholinergic chemosensory brush cells that are also present in this epithelium (Schütz et al., 2015).

FIGURE 4 | amiloride were added under continuous flow of Tyrode III into the chamber, so that indicated concentrations were reached initially and then washed out. In case of NaCl, concentration changes on top of the baseline of concentrations in Tyrode III are indicated. Thus, the total sodium concentration after addition of 1–150 mM ranged from 146 to 295 mM. Y-Axis depicts arbitrary units (AU) of Calcium Orange® fluorescence recorded by confocal laser scanning microscopy, correlating to [Ca2+] i . (A) NaCl evokes an increase in [Ca2+] i ; shown are mean and SEM. (B) Representative recording of changes in Calcium Orange® fluorescence in a single cholinergic (eGFP+) UBC in response to increasing concentrations of NaCl (blue arrowhead) and mannitol (orange arrowhead). (C) Depicted are peak values after stimulation with mannitol (150 mM, N = 22) and NaCl (1 mM, N = 22; 10 mM, N = 22, 50 mM, N = 24, 100 mM, N = 23; 150 mM, N = 21); \*P < 0.05, \*\*P < 0.01, \*\*\*P < 0.001 compared to mannitol 150 mM, Kruskall-Wallis test followed by Dunn's Multiple Comparison Test. (D) Representative recording of changes in Calcium Orange® fluorescence in a single cholinergic (eGFP+) UBC in response to NaCl (50 mM) in absence and presence of increasing concentrations of amiloride (0.01–10µM). (E) Depicted are peak values after application of NaCl (50 mM) in absence and presence of increasing concentrations of amiloride (0.01µM, N = 5; 0.1µM, N = 5; 1µM, N = 9; 10µM, N = 8; 100µM, N = 17), p-values were calculated by, Kruskal-Wallis test and are indicated in the figure. (F) Dose–inhibition curve of amiloride in UBC. IC50 = 0.47µM. (G) Representative recording of changes in Calcium Orange® fluorescence in a single cholinergic (eGFP+) UBC in response to NaCl (50 mM) in absence and presence of amiloride (0.1 mM). (H) Depicted are peak values after application of NaCl (50 mM, N = 17), amiloride (0.1 mM, N = 17), NaCl (50 mM) together with amiloride (0.1 mM, N = 17), and repetitive NaCl (50 mM, N = 10); \*P < 0.05, \*\*P < 0.01, \*\*\*P < 0.001, Kruskal-Wallis test followed by Dunn's Multiple Comparison Test.

Two out of 69 TRPM5-positive cells expressed Scnn1a-tdTomato (**Figures 3C,D**).

Among non-umbrella cells with nuclei located in deeper layers of the urethral epithelium, we detected co-localization of TRPM5-immunoreactivity and Scnn1a-tdTomato signal (30 of 46 TRPM5-positive cells, 65%, N = 3 animals) (**Figures 3A,B**) as well as TRPM5-positive cells without Scnn1a-tdTomato signal (**Figure 3B**). These observations support our findings in RT-PCR experiments and single cell sequencing that a subpopulation of UBC expresses α-ENaC.

Functionally, application of NaCl evoked significant increases in [Ca2+]i in 70% of the isolated cholinergic UBC. At concentrations of 50, 100, and 150 mM NaCl, but not at 1 or 10 mM, significant increases in [Ca2+]i were observed (**Figures 4A–C**). There was a tendency toward a decline in the evoked increase in [Ca2+]i with increasing NaCl concentration. To test for a possible osmolarity effect, increasing concentrations of mannitol were administered alternating with corresponding concentrations of NaCl (**Figure 4B**). Since mannitol had no stimulatory effect upon [Ca2+]i even at 150 mM (**Figure 4B**), the observed reaction of UBC to 50 mM NaCl, which was used for further characterization of polymodality, is not an osmolarity effect. Amiloride, an ENaC inhibitor that also suppresses salt perception in type I taste cells of the taste buds (Vandenbeuch et al., 2008), fully blocked the [Ca2+]i response in UBC to NaCl (50 mM) with an IC50 of 0.47µM (**Figures 4D–F**). Even at the highest concentration used (100µM), the inhibitory effect of amiloride was reversible upon wash-out (**Figures 4G,H**). Thus, the NaCl-induced increase in [Ca2+]i in UBC is amiloridesensitive. This, however, does not seem to involve the canonical αβγ-ENaC which was shown to detect low concentrations of sodium in taste receptor cells of fungiform papillae in mice (Chandrashekar et al., 2010). First, only 1/6 UBC expressed all three ENaC subunits detected by NGS. Second, activity of mouse αβγ-ENaC appears to be maximal at 60 mM extracellular sodium (Sheng et al., 2004), a concentration which is below the stimuli used in the present study (≥50 mM NaCl added to Tyrode III). Third, this response to the NaCl stimulus was sensitive to amiloride, whereas baseline [Ca2+]i in UBC (in the presence of 145 mM Na+ in Tyrode III) was not (**Figure 4G**). Fourth, even though this study used calcium-imaging and this does not directly measure ENaC-activity, the IC50 for the observed inhibition of the calcium signal by amiloride is above that reported for inhibition of mouse αβγ-ENaC (0.1µM) (Ahn et al., 1999). This might suggest an alternative amiloride-sensitive cation channel containing the ENaC α-subunit. Recently, it was shown that α-ENaC can assemble with alternative ion channels such as the acid sensing ion channel 1 (Jeggle et al., 2015), which may form a non-selective cation channel (Trac et al., 2017). The expression of acid sensing ion channels in UBC was, however, low and inconsistent (**Figure S1**). Alternatively, α-ENaC can form homomeric ion channels in vitro (Canessa et al., 1994b). Their physiological function, however, remains to be proven in vivo.

Irrespective of the molecular composition of the amiloridesensitive sodium conductance in UBC, exposure to high concentrations of NaCl might trigger a membrane depolarization which may stimulate calcium-influx via voltage-gated calcium channels and subsequent release of acetylcholine. UBC showed consistent expression of α-subunits of the L-type voltagegated calcium channels Cav1.2 (Cacna1d), Cav1.3 (Cacna1d), and Cav2.3 (Cacnac1e) (**Figure S2**). Furthermore, there was strong expression of the auxiliary subunit β4 (Cacnb4) in 5/6 cells. The auxiliary β-subunits are generally important for membrane expression and the β4-subunit seems to determine subcellular membrane-localization in polarized cells (Campiglio and Flucher, 2015). Voltage-gated calcium channels thus represent promising targets for the coupling of NaCl-induced membrane depolarization to acetylcholine release in UBC.

The physiological meaning of salt responsiveness of cholinergic UBC remains uncertain. In adult C57BL/6J mice, urinary sodium concentration is around 150 mM, similar to our 145 mM baseline in Tyrode buffer, and can significantly increase during water deprivation or high-salt intake (Li et al., 2012). During such conditions, cholinergic UBC may thus be exposed to sodium concentrations which trigger calcium responses as shown in this study. UBC are interpreted as sentinels of the lower urinary tract equipped for monitoring the mucosal surface for potential hazardous content, especially bacterial products (Deckmann et al., 2014; Deckmann and Kummer, 2016; Kummer and Deckmann, 2017). Threatening bacterial infections, however, are usually not connected to increased salt concentrations. Thus, α-ENaC may here serve other functions than monitoring luminal NaCl concentration. Canonical ENaC holds a key position in maintaining electrolyte

and water homeostasis, e.g., concentration of primary urine in the kidney (Kellenberger and Schild, 2002). Given the low number of cholinergic UBC in the urethra and their minimal exposure to the luminal surface, this function appears rather unlikely for this particular cell type. However, ENaC is also a mechanosensitive ion channel, reacting to shear stress (Althaus et al., 2007; Guo et al., 2016). This opens the possibility that ENaC-subunit carrying UBC may be involved in sensing urine flow in the urethra. Notably, as mechanical strain affects the entire epithelium and is not restricted to the luminal membrane, it will reach UBC without a clear connection to the luminal surface ("closed type," see **Figure 3B** and Deckmann and Kummer, 2016). Cholinergic UBC are connected to sensory nerve fibers and, reflexively, initiate micturition in response to a bitter stimulus in the urethral lumen (Deckmann et al., 2014). This has been interpreted as a protective reflex in that potentially hazardous content will be flushed out (Deckmann et al., 2014; Kummer and Deckmann, 2017). Voiding efficiency is augmented by sensory feedback from the urethra, where flow sensors are physiologically well characterized but not yet defined anatomically (Todd, 1964; Peng et al., 2008; Danziger and Grill, 2015). Thus, mechanosensitivity of cholinergic UBC may serve to augment the reflex response they have initiated.

To test for polymodal properties, cholinergic UBC were successively exposed to NaCl and ATP (N = 90; 70% responded to NaCl), to NaCl and denatonium (N = 36; 67% responded to NaCl), and to all three stimuli (N = 37; 65% responded to NaCl, **Figure 5**). When responses to both NaCl and denatonium were tested on 36 UBC, all three possible response patterns occurred in a balanced distribution (**Figure 5**): 42% NaCl only, 33% denatonium only, 25% both stimuli. These percentages are roughly reflected by the (immuno)histochemical (65% of UBC expressing Scnn1a-tdTomato signal) and by the NGS data with 4/6 cells (67%) expressing Scnn1a, and 2 of them (33%) expressing additionally a known receptor for denatonium, i.e., Tas2r108 (**Figure 2**). Of course, the small total number of cholinergic UBC subjected to NGS (N = 6) precludes a systematic quantitative analysis.

We have previously shown that a substantial number of denatonium-responsive UBC also reacts to monosodium glutamate (Deckmann et al., 2014). In terms of oropharyngeal gustation, these substances reflect an aversive (denatonium: bitter) and an attractive (monosodium glutamate: umami) stimulus, and, accordingly, are perceived by distinct cell populations, which still are considered as subtypes of type II taste cells (Chaudhari and Roper, 2010). The present data show an even broader diversity of UBC properties in that some of them share features also with type I cells of taste buds, expressing ENaC and being responsive to NaCl (Vandenbeuch et al., 2008). These findings further substantiate the polymodal character of cholinergic UBC. As far as further distinctive criteria are missing, we interpret the multiple combinations of responsiveness to various chemosensory stimuli and gene expression of related signaling components as phenotypic variation of a broadly tuned, polymodal chemosensory cell rather than defining multiple, clearly separated cell types.

### CONCLUSION

In sum, we could show that a fraction of cholinergic UBC expresses α-ENaC and responds to the salty stimulus NaCl in an amiloride-sensitive manner. This feature does not define a new subpopulation of UBC, but rather emphasizes their polymodal character.

### AUTHOR CONTRIBUTIONS

KD designed research and performed statistical analysis. KD, CK, PatS, PauS, MK, and SO performed research and analyzed data.

### REFERENCES

KD and WK obtained funding. KD, WK, AP, and MA drafted the manuscript. Work was supervised by WK and KD.

### FUNDING

This work was supported by a University Hospital of Giessen and Marburg (UKGM)-Justus-Liebig-University (JLU)-Cooperation Grant (# 7/2016 GI to KD), the German Research Foundation (KU 688/8-1 to WK), and the Else Kröner-Fresenius-Stiftung (2016\_A90 to KD).

### ACKNOWLEDGMENTS

We thank M. Bodenbenner, T. Eiffert, and K. Michael for skillful technical assistance as well as the FACS Core Facility of the University of Giessen for technical support. We also thank J. Staiger and J. Guy for providing material.

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcell.2018. 00089/full#supplementary-material

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Kandel, Schmidt, Perniss, Keshavarz, Scholz, Osterloh, Althaus, Kummer and Deckmann. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# In silico Description of LAT1 Transport Mechanism at an Atomistic Level

Luca Palazzolo1 , Chiara Parravicini 1 , Tommaso Laurenzi 1 , Uliano Guerrini 1 , Cesare Indiveri 2 , Elisabetta Gianazza1 and Ivano Eberini 1 \*

1 Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, Milan, Italy, 2 Dipartimento di Biologia, Ecologia e Scienze della Terra, University of Calabria, Cosenza, Italy

The molecular mechanism of transport mediated by LAT1, a sodium-independent antiporter of large neutral amino acids, was investigated through in silico procedures, specifically making reference to two transported substrates, tyrosine (Tyr) and leucine methyl ester (LME), and to 3,5-diiodo-L-tyrosine (DIT), a well-known LAT1 inhibitor. Two models of the transporter were built by comparative modeling, with LAT1 either in an outward-facing (OF) or in an inward-facing (IF) conformation, based, respectively, on the crystal structure of AdiC and of GadC. As frequently classic Molecular Dynamics (MD) fails to monitor large-scale conformational transitions within a reasonable simulated time, the OF structure was equilibrated for 150 ns then processed through targeted MD (tMD). During this procedure, an elastic force pulled the OF structure to the IF structure and induced, at the same time, substrates/inhibitor to move through the transport channel. This elastic force was modulated by a spring constant (k) value; by decreasing its value from 100 to 70, it was possible to comparatively account for the propensity for transport of the three tested molecules. In line with our expectations, during the tMD simulations, Tyr and LME behaved as substrates, moving down the transport channel, or most of it, for all k values. On the contrary, DIT behaved as an inhibitor, being (almost) transported across the channel only at the highest k value (100). During their transit through the channel, Tyr and LME interacted with specific amino acids (first with Phe252 then with Thr345, Arg348, Tyr259, and Phe262); this suggests that a primary as well as a putative secondary gate may contribute to the transport of substrates. Quite on the opposite, DIT appeared to establish only transient interactions with side chains lining the external part of the transport channel. Our tMD simulations could thus efficiently discriminate between two transported substrates and one inhibitor, and therefore can be proposed as a benchmark for developing novel LAT1 inhibitors of pharmacological interest.

Keywords: LAT1, targeted molecular dynamic, aminoacid transporters, tyrosine, molecular docking

### INTRODUCTION

The L-type amino acid transporter 1 (LAT1, SLC7A5) is a ubiquitous Na+- and H+-independent antiporter, involved in cellular uptake of essential amino acids. Being over-expressed in many human cancers that are characterized by an increased demand of amino acids, it was recently acknowledged as a novel target for cancer therapy (Haase et al., 2007; Napolitano et al., 2017b). LAT1 is also expressed in the blood-brain barrier (BBB), where it allows the transit

Edited by:

Laszlo Otvos, Olpe LLC, United States

#### Reviewed by:

Francisco Solano, Universidad de Murcia, Spain Sam P. De Visser, University of Manchester, United Kingdom

> \*Correspondence: Ivano Eberini<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Chemical Biology, a section of the journal Frontiers in Chemistry

Received: 02 February 2018 Accepted: 25 July 2018 Published: 24 August 2018

#### Citation:

Palazzolo L, Parravicini C, Laurenzi T, Guerrini U, Indiveri C, Gianazza E and Eberini I (2018) In silico Description of LAT1 Transport Mechanism at an Atomistic Level. Front. Chem. 6:350. doi: 10.3389/fchem.2018.00350

**291**

of essential amino acids and also of some drugs, including the antiparkinson drug L-dopa and the anticonvulsant gabapentin (Dickens et al., 2013; Scalise et al., 2018).  For these reasons, much attention is currently devoted to LAT1 transporter activity (Napolitano et al., 2017a,b; Scalise et al., 2018), and to its inhibition, e.g., with 3,5-diiodo-L-tyrosine (DIT), fenclonine, and acivicine (Geier et al., 2013; Schlessinger et al., 2013).

Like other members of the amino-acid-polyamineorganocation (APC) family (Napolitano et al., 2017a), LAT1 is characterized by 12 transmembrane α-helices, which can be grouped in three different domains (Boudker and Verdon, 2010; Diallinas, 2014): α-helices 1–5 and 6–10 are involved in the transport mechanism and have an antiparallel symmetry in their secondary structures, while 11 and 12 are necessary for the homo-dimerization of the transporter. The repetitive symmetry of the first two domains gives to the α-helices a discrete mobility within the cell membrane and defines the binding site for transported substrates, in the middle of the transport channel. This structural feature is defined "LeuT fold," from the name of the first crystallized transporter belonging to the APC family (Krammer et al., 2016; Kazmier et al., 2017). For LeuT fold proteins, a model of transport has been proposed: in the initial phase of the process, the protein lies in a "outward-facing" (OF) conformation and receives the transported substrate in its binding site. The interaction between protein and transported substrate promotes the gate closure: this starts from the rotation of a specific conserved aromatic amino acid (see below), which in turn leads to a conformational change in the whole protein, promoting its transition to an "inward-facing" (IF) conformation (Gao et al., 2010; Krammer et al., 2016). Afterward, the substrate is released by the transporter in the intracellular space and the protein, with a new substrate to be brought to the extracellular space, returns to its OF conformation, passing through intermediate transitory occluded states, as shown in **Figure 1**.

The details of this mechanism are not yet clarified, as the few APC crystallographic structures so far resolved are either in OF (AdiC, LeuT, and BetP) or in IF (Mhp1 and GadC) conformations, and none is in a non-occluded/occluded intermediate state (Singh et al., 2008; Weyand et al., 2008; Quick et al., 2009; Ressl et al., 2009; Zhou et al., 2009; Gao et al., 2010; Kroncke et al., 2010; Shimamura et al., 2010; Kowalczyk et al., 2011; Perez et al., 2011; Ma et al., 2012; Koshy et al., 2013; Simmons et al., 2014; Ilgü et al., 2016). In this context, molecular modeling, based on comparative modeling together with non-standard molecular dynamics (MD) simulations, such as targeted molecular dynamics (tMD), steered molecular dynamics (sMD) or meta-dynamics (MTD), has been widely exploited to shed light on the APC transport mechanism. For instance, in AdiC, an arginine/agmatine antiporter of E. coli, the interactions between the transported substrate and the side chains that define the transport channel have been studied in depth, through non-standard molecular dynamics

simulations (Krammer et al., 2016), to identify the pivotal residues in the process (Shaikh and Tajkhorshid, 2010; Cheng and Bahar, 2013, 2014; Geier et al., 2013; Zhao and Noskov, 2013; Stolzenberg et al., 2015; Krammer et al., 2016; Chiu et al., 2017).

For LAT1, a specific phenylalanine (Phe252) has been proposed as the "upper gate," since its closure is involved it the transition from OF to IF.

Despite its biochemical and pharmacological interest, LAT1 structure has not been solved yet by crystallography. Some LAT1 comparative models have already been proposed, mostly based on the crystallographic structure of AdiC in the OF conformation (Dickens et al., 2013; Geier et al., 2013; Augustyn et al., 2016; Colas et al., 2016; Napolitano et al., 2017a; Ilgü et al., 2018) which covers both the open and closed LAT1 conformations. However, since these models are all based on the only available OF conformation of different AdiC crystallographic structures, it is currently unknown which residues are actually involved in the substrate transport when LAT1 is in its IF conformation.

To overcome this limitation, in the present work we performed 12 different tMD simulations, involving two transported substrates, tyrosine (Tyr) and leucine methyl ester (LME), and a transport inhibitor, 3,5-diiodo-L-tyrosine (DIT) (Geier et al., 2013; Zur et al., 2016), and discussed our findings. With this investigation, we meant: (i) identifying the key residues for the transport along the whole channel, (ii) comparing the transport mechanism of molecules characterized by opposite functional effects on LAT1 and (iii) providing the first in silico evidence of a LAT1 inner gate to guide further in vitro experiments.

### MATERIALS AND METHODS

### Comparative Modeling of LAT1

LAT1 comparative modeling was based on already published multiple alignments (Napolitano et al., 2017a,b) of some members of the APC superfamily, including three amino acid/poly-amine antiporters [AdiC, CadB and PotE], one cationic amino acid transporter (CAT) [CAT6], one amino acid/choline transporter (ACT) [Uga4], one glutamate/GABA antiporter (GGA) [GadC], LAT1 and LAT2 (see **Supplementary Material**). Multiple alignment was carried out by the program Clustal Omega (Li et al., 2015).

According to their crystallization state, the crystallographic structures of AdiC (PDB ID: 3OB6), with Trp202 gate residue in an open conformation, and GadC (PDB ID: 4DJI) were selected as templates for modeling LAT1 OF and IF conformations, respectively. Selected 3D structures were first optimized and refined for further computational steps by correcting crystallographic-related errors and/or by filling up any unresolved residues (e.g., some amino acids in the GadC C-terminus) through the MOE Structure Preparation Module of the Molecular Operating Environment 2016.08 (MOE, Chemical Computing Group, Montreal, Canada). Comparative models were produced with the MOE Homology Model program with default settings, using the Amber10:EHT force field. Ten intermediate models were built and refined for each LAT1 conformation. Final models were selected according to the electrostatic solvation energy calculated with the Generalized Born/Volume Integral (GB/VI) methodology. The Ramachandran plot, the side chain packing, and the stereo chemical quality of the selected structures were checked with the MOE Protein Geometry module in order to verify that all these parameters were consistent with typical values found in crystal structures.

### Molecular Dynamics Simulation

The Desmond Molecular Dynamics System (D. E. Shaw Research, New York, NY, 2018, Schrödinger, New York, NY, 2018) system builder tool was used to place the OF LAT1 model into a POPC membrane bilayer. LAT1 orientation was set up according to the OPM server (Lomize et al., 2012), which provides spatial arrangements of membrane proteins with respect to the hydrocarbon core of the lipid bilayer. The Nand C-termini of the protein were capped. The system was solvated with 10,174 TIP3P water molecules in a cubic box with 90 Å edges; the exceeding positive charge was neutralized by adding chloride ions; sodium chloride was added up to 0.1 M concentration. The system was energy-minimized down to a root mean square (RMS) gradient of 0.05 kcal/mol/Å2 using the MOE software, to relax the system and remove clashes between protein, membrane and solvent in the new setup.

To produce an equilibrated model of OF LAT1, the system was submitted to a 150 ns molecular dynamics simulation (MD) using NAMD, prepared through the MOE graphical interface (Phillips et al., 2005), under periodic boundary conditions (PBC). The following parameters were set: 300 K and Langevin thermostat for temperature coupling, 1 bar and Nosé-Hoover Langevin piston for pressure coupling, 2 fs as integration time step. Coordinates and velocities of each atom were saved every 1 ps. The Amber10:ETH force field with the reaction-field treatment for electrostatics was applied in all the computational procedures. Analyses of the trajectories were performed with the VMD tool (Humphrey et al., 1996).

### Targeted Molecular Dynamics Simulations

Targeted molecular dynamics (tMD) is a method to observe large-scale transitions between two known end-point conformations of a molecule, initial and final (target). To this purpose, a restraint energy term is added to the total energy of the system: frame-by-frame, each specified atom is subject to a harmonic potential (i.e., a spring) that pulls it toward its target position. The restraint energy term is assumed to be proportional to the square the difference between (a) the RMSD from instant to final coordinates and (b) the RMSD that there would be from instant to final coordinates assuming a linear transition between the initial and target coordinates. As with a harmonic oscillator, a constant of proportionality k (i.e., spring constant) expresses how strong is the force with which the system is pulled toward its target coordinates (Schlitter et al., 1994).

The LAT1 model coming from the equilibrating MD phase was energy minimized and used as the starting state, whereas the final state was generated by replacing the OF LAT1 model embedded into the membrane with the IF model.

Transported substrates and inhibitor were added to the systems at the top of the simulation box in the starting states, aligning them along the Z-axis of the transport channel. The solvent bulk was then energy-minimized down to a RMS gradient of 0.05 kcal/mol/Å2 , using the MOE software and keeping protein, substrates/inhibitor and membrane atoms fixed.

To preserve the optimized atomic configuration coming from the model based on AdiC structure, only the α-carbon positions of LAT1 and the atoms of substrates/inhibitor were set as targeted atoms to guide the OF/IF transition, while side chains were set as freely moving.

A total of 12 different tMDs were performed, four with Tyr, four with LME and four with DIT. Performed tMD simulations and their identifiers are listed in **Table 1**. In detail, for each investigated compound the same tMD simulations were carried out under the following conditions: (i) spring constant k = 100 for 10 ns, (ii) sequential 5 ns-tMD with decreasing values of k (k = 90, 80, and 70). The spring constant k controls the magnitude of the elastic potential that pulls each atom toward its final coordinates (Phillips et al., 2005). A schematic flowchart of the in silico set up is shown in **Figure 2**.

The following parameters were set: 300 K and Langevin thermostat for temperature coupling, 1 bar and Nosé-Hoover Langevin piston for pressure coupling, 2 fs as integration time step. Coordinates and velocities of each atom were saved every 0.5 ps. Amber10:ETH was set as force field for all tMD simulations. All the tMD simulations were carried out using NAMD and prepared through the MOE graphical interface (Phillips et al., 2005).

### tMD Analyses

Aqua-Duct (v. 0.3.7 - http://www.aquaduct.pl) was used to analyze the trajectories of the substrates/inhibitor across the transporter. For our calculations, we tracked every molecule passing through the LAT1 channel, defined as a spherical zone of radius 6 Å, centered on residues Thr109, Val190, Asn273, Leu385, and Phe451. All the trajectories were also analyzed using GROMACS analysis tools, supported by the VMD plugins. Trajectory time frames were clustered on the basis of Tyr, LME, and DIT positions relative to the LAT1 transport channel according to the simple linkage methodology. Reported data

refer to the centroids of representative clusters. In order to compute the contribution of individual residues to the total interaction energy, a MOE svl script based on the interaction force fingerprint (IFFP) was applied. This method partitions the native force-field potentials and derives the residue-specific interaction forces of the ligand-receptor complex characteristic of hydrogen bonding, steric clashes, and other aspects of molecular interactions. The sum of these forces per residue provides the overall interaction of each single amino acid with the ligand and allows to compute the total interaction energy associated to the complex. It specifically works by subtracting non-bonded interactions of the ligand with each amino acid residue and using the resulting force vectors to describe the slope of the remaining potential (Shadnia et al., 2009).

### Molecular Docking Simulations

Resulting tMD trajectories were clustered, and the representative conformations (i.e., centroids of representative clusters) were used as LAT1 structures for molecular docking simulations (Jurik et al., 2015; Colas et al., 2016).

The binding sites of representative conformations were identified through the MOE Site Finder program. Molecular docking simulations of both substrates and inhibitor were carried out with the MOE Docking program with default settings, as previously described (Galli et al., 2014). Briefly, a multi stage docking framework was applied, according to the MOE Docking algorithm, which includes conformational search, placement, initial scoring, refinement and final scoring. The protein was treated as a rigid body, while intermolecular bonds of docked

FIGURE 2 | Flowchart of in silico set up. LAT1 OF and IF models were built starting from AdiC and GadC crystal structures, respectively, via homology modeling. LAT1 OF model was equilibrated through a 150 ns MD simulation and then used as starting structure for all the tMD simulations. α-Carbons of LAT1 IF model were used as targets for all the tMD simulations.

compounds were allowed freely rotation to generate preferred conformations prior to the placement stage. The Amber10:EHT force field was applied for both protein and substrates/inhibitor parametrization.

Depending upon the docking stage, docked compounds were ranked according to two different empirical scoring functions which provide an estimation of the free energy of binding (1G) and include in their calculation, among others contributions, solvation and entropy terms, or enthalpy terms based on polar interaction energies. In detail, the London dG scoring function and the forcefield-based GBVI/WSA 1G scoring function were used for the initial and the final scoring of the docking poses, respectively (Naïm et al., 2007; Labute, 2008). More negative values of this parameter, expressed in kcal/mol, indicate a gain, less negative values a worsening in affinity for the substrate (Sensi et al., 2014; Platonova et al., 2017).

### In silico Site-Directed Mutagenesis

Single point mutations were generated with the MOE Residue Scanning tool; the selected residues were mutated to alanine. To obtain more accurate results, the sites being mutated were treated as flexible by applying a conformational sampling method to both the target amino acid and the surrounding residues and generating an ensemble of conformations. Then, for each analyzed property of the protein ensemble monitored through the conformational search (i.e., protein thermostability and binding affinity for the substrate), average values were computed using a Boltzmann distribution. In detail, the Low Mode MD sampling method was selected for each in silico mutation to generate 25 conformational ensembles and to sample interactions between substrate and surrounding residues. To account for the effect of mutation on Tyr transport, relative thermostability (1Stability) and binding affinity (1Affinity), with respect to the wild type LAT1, were computed as the Boltzmann average of the relative stabilities/affinities of the ensemble.

The change in protein stability due to mutation was computed through the MOE protein stability scoring function, trained on over 3000 single point mutations taken from the FoldX (Guerois et al., 2002) and PoPMuSiC-2.0 (Dehouck et al., 2009) datasets. This method is based on the prediction of the difference in stability (s) between the mutant and wild type in the folded (f) and unfolded (u) states of the protein, expressed as 11Gs,computed as follow:

$$
\Delta\Delta G\_{\sharp} = \Delta G\_{f}^{Mut} - \Delta G\_{f}^{WT} = \Delta G\_{sf}^{WT \to Mt} - \Delta G\_{su}^{WT \to Mt}
$$

Accordingly, a model based on Linear Interaction Energy is applied, in which 1G can be expressed as the change in the residue environment interaction energy going from wild-type to mutant, where the interaction energy is the change between the energy of the protein and the energy of the protein without the residue of interest.

The GBVI/WSA dG scoring function, an AMBER/GBVIbased scoring function trained on 99 protein-ligand complexes (Corbeil et al., 2012), was used to calculate the differences in affinity for the transported solute substrate Tyr upon mutations.

More negative values of 1Stability and 1Affinity, expressed in kcal/mol, indicate increased stability of the transported structure and gain of affinity for the substrate. Conversely, more positive/less negative values of both parameters indicate loss in complex stability and worsening of affinity.

### RESULTS AND DISCUSSION

### OF and IF LAT1 Models

LAT1 comparative models were built from multiple alignments of a set of transporters belonging to the APC family, as previously described (Napolitano et al., 2017a) and reported in **Supplementary Material**. Despite the existence of a binding partner of LAT1, SLC3, most of the published wet transport data have been obtained on the monomeric form of LAT1 (Napolitano et al., 2017a), which was demonstrated to exert its activity also in this form. For this reason, to characterize the transport mechanism of LAT1, we focused only on its monomeric form. In order to build LAT1 models in two different conformations, the crystallographic structures of known APC transporters were analyzed and compared by structural superposition.

Making reference to our sequence alignment (Napolitano et al., 2017a), as reference structure to model LAT1 in its OF conformation we selected AdiC, which, even with a sequence identity of 21% and similarity of 34%, represents the most suitable template available for modeling LAT1.

Membrane proteins are hard targets for structural biology due to the intrinsic instability of their crystals. Despite the number of solved structures has exponentially grown in the last few years, comparative modeling is still the method of choice if the unknown protein shares any significant sequence similarity with an experimental structure. However, the identification of topological fingerprints and sequence similarity networks has proven useful for improving the accuracy of low- (<30% sequence identity) or mid-range (<50%) comparative models (Schlessinger et al., 2013). In this respect, as already done for GPCRs (Isberg et al., 2016), also for some membrane transporters a numbering scheme has been proposed, providing common reference points within the topology, useful for guiding homology modeling and structural comparison across the entire family (Lee et al., 2016).

Among the few homologous transporters solved so far in IF state, namely ApcT and GadC (Shaffer et al., 2009; Ma et al., 2012), the latter shows a more clearly developed IF conformation than ApcT. This conclusion is supported by the higher RMSD values obtained when AdiC and GadC are superposed by structural alignment (6.1 Å), compared to those obtained by superposing AdiC and ApcT (4.9 Å). Moreover, α-helix 1, which is involved in the conformational changes in the transition from the OF to the IF state, is fully inward open in GadC (**Supplementary Figure 1**). On this basis, despite the low sequence identity, GadC was selected as the most suitable template to model LAT1 in IF conformation. However, this model was not used in any tMD simulations, but just to define the reference final (target) position of the α-carbons.

Protein geometry has been checked for the two crystallographic structures used as templates, and for the OF and IF models. The checked parameters are not significantly different between experimental (**Supplementary Figures 2A,B**) and computational structures (**Supplementary Figures 2C,D**).

The structural comparison between the two models allows pointing out the regions with high mobility and the ones less involved in conformational changes during the transport cycle. The RMSD between OF and IF conformation amounts to approximately 7.5 Å overall and is color coded on a residue-toresidue basis in **Figure 3A**. In agreement with previous literature data on homologous transporter structures (Krammer et al., 2016), the α-helices 1, 4, 11, and 12 of our models show a high RMSD value (average 6.5 Å) along their whole length; α-helices 2, 6, and 7 also show a high RMSD value (average 6.8 Å), but this is associated to conformational rearrangements in only a portion of their length, suggesting that the α-helices are involved to a different extent in this structural transition.

Two different transport channel surfaces were identified in OF (**Figure 3B**) and IF (**Figure 3C**) LAT1 models. In particular, in the OF model the transport channel surface is open to the extracellular side of the membrane; conversely, it is closed in its lower portion, mainly by residues Tyr259, Thr345, and Asn258 (**Supplementary Figure 3A**). On the other hand, the IF structure has a well-defined channel, open to the intracellular side of the membrane; the main residues that close the upper portion of the channel are Ile147, Phe252, Ser143, and Phe69 (**Supplementary Figure 3B**).

To ensure the robustness of our procedure, the stability of the OF model, used as reference structure for the following steps, was carefully assessed via 150 ns of MD simulation. To this end, the system was relaxed and the OF LAT1 structure equilibrated in an explicit membrane bilayer. Besides protein geometry quality validation, the general stability of the OF LAT1 model is also confirmed by the short-simulated time necessary for the protein to reach a RMSD plateau, characterized by a value (<3 Å) typical of stable proteins (**Supplementary Figure 4**). As expected, the largest root mean square fluctuation (RMSF) computed for the α-carbons is associated with the C-terminus (residues 437-441), a typical protein end-effect. The extracellular loops, residues 117-119 and 175-180, are associated to a RMSF of approx. 4 Å. Conversely, α-helices RMSF is < 1 Å, suggesting very low flexibility for the transmembrane barrel structure. Our MD simulation thus suggests that the structure obtained from this equilibration phase is stable and it is also equivalent to the OF LAT1 structure already published in our previous works (Napolitano et al., 2017a,b) in which we validate our OF LAT1 model via in vitro experiments.

For these reasons, the equilibrated OF LAT1 model represents a suitable starting point for tMD simulations, supporting its use as a reference structure to guide LAT1 from an OF to an IF conformation.

### Differences in Substrates and Inhibitor Transport Mechanisms With k = 100

Frequently, classical MD simulations do not efficiently explore the conformational space and fail to reproduce large functional protein rearrangements. To bypass this limitation, tMD simulations were carried out. To model the transition from OF to IF LAT1 conformation within this protocol, the α-carbon

positions of the IF LAT1 model were taken as reference, disregarding the side chains; this selection avoids any potential issue connected with the use of a distant homologous template. Accordingly, IF LAT1 model was never used in any of our tMD simulations.

The use of different experimental setups in tMD allowed us to discriminate the ability of the investigated compounds (Tyr, LME and DIT) either to be transported or to inhibit LAT1 activity, as well as to capture the complete transition from OF to IF resulting in the passage of substrates across the transport channel.

In detail, a simulation at a the relatively high spring constant value (k = 100 for 10 ns) was useful to assess the whole transport cycle and to allow the passage of all substrates/inhibitor through the LAT1 channel; decreasing k values (90, 80, 70, for 5 ns) were set to identify the conditions at which transport is allowed or blocked for substrates and inhibitor and to appreciate differences in their transport mechanism (**Table 1**).

**Supplementary Movie 1** shows the entire transport simulation of the three compounds. Their paths during the tMD simulations were traced and are reported in **Supplementary Figure 5**. This analysis shows that, at the highest spring constant value, Tyr, LME and DIT are able to pass through the LAT1 transport channel, moving from the extracellular to the intracellular side of the membrane.

In these simulations, RMSF values computed for LAT1 αcarbons (**Figure 4A**) point out highly flexible regions, mainly corresponding to α-helices 1, 4, 6, 7, and 9. α-helices 11 and 12, required for LAT1 homo-dimerization (Napolitano et al., 2017a), are associated instead with very low local flexibility in T1 and D1 simulations, while α-helix 11 shows an increased flexibility in L1 simulation. As already mentioned above, the C-terminus shows the highest mobility.

Substrates/inhibitor path analysis does not provide any timedependent information on transport; for this reason, the Z-axes distances between substrates/inhibitor and Arg141 were plotted for the tMD simulations. Arg141 was selected as reference, since it is placed in the middle of the transport channel, on α-helix 3, which presents the lowest RMSD between the starting OF and final IF structures. Arg141 itself is associated to the lowest RMSF value, suggesting that this residue encounters minor changes during OF → IF transitions and thus represents a good reference to trace substrates/inhibitor movements along the transport channel.

Comparing protein RMSD (**Figure 4B**) with Arg141 distance plots (**Figure 4C**), it appears that OF → IF LAT1 transition (between 2 and 4 ns) occurs before the transit (from 5 ns) of transported substrates/inhibitors close to the gate (**Figures 3C**). These data suggest that transported substrates can be delivered to the intracellular side only after the transporter structural rearrangement starts taking place. Summing-up, these tMD simulations, run in an artefactual setup (high spring constant), demonstrate a faster transport for substrates than for DIT. As shown in **Figure 4C**, Tyr and LME reach the binding site, near the Phe252 gate, approx. 1 ns before DIT, and remain there for 2 ns. Afterwards, transported substrates move down the second half of the transport channel, reaching the intracellular side in approximately 8 ns. Conversely, DIT reaches the binding site within 6 ns, stops there for about 2 ns, then exits very quickly from the transport channel. The distance profile analysis lets us hypothesize the existence of a second gate, other than Phe252, as described in AdiC (Krammer et al., 2016), which blocks the transport of the substrates until a suitable LAT1 rearrangement occurs.

In order to identify this putative gate, a cluster analysis was performed; results are reported in **Table 2**. Data discussed in

detail in the text only refer to the centroids of the most populated clusters.

In all the 10 ns tMD simulations with k = 100, the three main clusters represent an initial, an intermediate and a final state of the transport, in which substrates and the inhibitor are at the extracellular side, buried in the channel and at the intracellular side of LAT1 channel.

For T1 the most populated cluster is C1; observing Tyr interactions with surrounding residues, it is possible to identify a putative inner gate, composed of three amino acids: Phe262, Arg348, and Thr345.  As shown in **Figure 5A**, hydrogen bonds are formed between the amino group of Tyr and the hydroxyl group of Thr345, and a salt bridge between the carboxylic group of Tyr and the basic group of Arg348.

To evaluate whether the interactions between Tyr and the putative inner gate residues pointed out by tMD simulation are relevant, we used molecular docking to sample Tyr binding modes more accurately, using the Tyr coordinates reported in C1 to address the binding site. We obtained five refined docking poses of Tyr, which had an RMSD ranging from 0.1 to 1.3 Å when compared to the Tyr binding mode observed in C1.

The top-scoring pose has an associated binding free energy of −7.4 kcal/mol and overlaps with the tMD reference pose with an RMSD value of 0.1 Å (**Figure 5B**), suggesting that C1 cluster is representative of an energetically-stable conformation of both substrate and transporter.

As already shown in our previous papers, a direct relationship exists between dissociation constant (Ki) computed from binding free energy on molecular docking poses and in vitro data. Indeed, it has been reported that the accuracy of in silico estimated Ki values is approx. one order of magnitude with respect to the experimental values (Eberini et al., 2008; Galli et al., 2014), implying that the accuracy of the binding free energy is approximately 1.3 kcal/mol.

The contribution of each interacting residue to LAT1 model stability and to its affinity for the substrate Tyr has been TABLE 3 | Interaction force fingerprint for simulations with k = 100.

TABLE 4 | In silico mutagenesis for C1 cluster. Mutations are sorted by differences in protein stability contribution.

estimated, with respect to the wild type, upon in silico cumulative mutations of each specified residue to alanine (**Table 4**). We found that the mutation of a single residue weakly affects the affinity for the transported substrate, whereas in cumulative mutations Arg348 seems to be the most relevant residue, among the investigated ones: the high 1Affinity value suggests that this amino acid may play a pivotal role in the interaction with the substrate.

Only by mutating Phe262, 1Stability was appreciably affected, showing an increase with the number of cumulative mutations. When Phe262 and two or more other residues are simultaneously mutated, the 1Stability value is higher than 3 kcal/mol (Tokuriki et al., 2008).

These site-directed mutagenesis results, even if limited by their merely in silico nature, could be suggestive for further in vitro experiments, aimed at validating the proposed LAT1 transport mechanism.

The same analysis was repeated for both L1 (LME) and D1 (DIT) tMDs (**Table 3**). In C5, LME is placed in proximity of the outer gate where it establishes a Pi-stack interaction with Tyr147 and forms a H-bond with Thr71 (**Figures 5C,D**). The gating residue Phe252 also contributes to the total interaction energy. In C8, it was possible to identify crucial interactions between DIT, which is buried into the channel, and two residues, Thr345 and Tyr259; according to IFFP these two residues

provide the highest contribution to the total interaction energy. Other residues, reported in **Figures 5E,F**, were recognized as important for the total interaction energy. C3, C6, and C9 clusters, representative of the final intracellular localization of transported substrates/inhibitor, and descriptive of the final states of dynamics, were not extensively analyzed since no interactions with LAT1 were observed.

In addition to the outer gate including Phe252, these tMD simulations suggest the presence of a second putative inner gate, located in the inward facing region of the transport channel; this involves Thr345, which interacts with Tyr and DIT. Through tMD it is possible to appreciate the different behavior of Tyr, LME and DIT since the substrates and the inhibitor transit through LAT1 with different timing.

### Decreasing Spring Constant tMD

Simulations of Tyr, LME, and DIT transport by LAT1 were repeated with k set at 90, 80, and 70 and results were processed through cluster analysis and IFFP as described above; an overview on the outcome is provided in **Tables 5**, **6**.

An extensive analysis of all the tMD trajectories and substrate paths (**Supplementary Figure 5**) shows that, as the spring constant k decreases, Tyr slips into LAT1 at different depths. Only in T2, Tyr completely crosses the membrane, exiting the LAT1 convex-hull. In T3, Tyr enters the transport channel, but does not exit the LAT1 convex-hull, whereas, in T4, Tyr is blocked just below the external gating region, Phe252. LAT1 rearrangements from OF to IF conformation occurring in T2, T3, T4 tMDs are shown in **Supplementary Movies 2**–**4**.

The initial position of Tyr in the simulated system is shown in Figure 6.A. RMSD and RMSF plots (**Figures 6B,C**), for T2, T3 and T4 tMD simulations, do not significantly differ. The distance between Tyr and Arg141 was plotted as a function of time for all the 5ns tMDs, in order to assess the time spent by Tyr in the different regions of the LAT1 transport channel. In **Figure 6**.D it is possible to distinguish some steps in which Tyr is temporarily trapped, allowing us to hypothesize ongoing protein rearrangements. Cluster analysis of Tyr position along the LAT1 transport channel (**Table 5**) and IFFP assessment of residue contribution to interaction (**Table 6**) show that in C11 Tyr takes contact with the previously identified residues, i.e., Thr345, Arg348. Under the weaker spring constant value Tyr259 turns out to be a key residue in the putative inner gate. Other aromatic residues, Phe244 and Phe262, line the transport channel and participate in interactions with transported substrates. Also in this case, molecular docking simulations could reproduce the Tyr binding mode within this specific cluster (not shown). We obtained four refined docking poses of Tyr with an RMSD from the C11 Tyr reference coordinates ranging from 0.2 to 1.8 Å, and only one docking pose had an RMSD above the value of 2 Å, that is taken as a cut-off to discriminate between different binding modes (Poli et al., 2016). The top-scoring pose has a binding free

energy of approx. −6.5 kcal/mol, associated with a RMSD of 0.2 Å, suggesting that the interaction mode between substrate and transporter pointed out by tMD simulation can also be carefully reproduced by molecular docking.

In C14, Tyr is close to Tyr259 and to Thr345, which are the main residues contributing to the IFFP. Interestingly, Phe252 is in an open conformation, whereas it is in a closed conformation in C15. The most populated cluster in T4 is representative of Tyr permanence in the extracellular side. C16 and C17 are representative of the Brownian movements of Tyr in the upper side, while C18 is representative of the last stage of the Tyr path; in this cluster, Tyr interacts with Thr345 and Tyr259. None of the 5 top-scoring poses obtained by docking simulation could reproduce the Tyr position in C18, suggesting that C18 is a transitory conformation of Tyr::LAT1 complex during the substrate transport.

From the analysis of the LME trajectories, the substrate reaches the intracellular space both in L2 and in L3. LME transport across LAT1 is shown in **Supplementary Movies 5**–**7**. The starting configuration of LME within the simulated system in tMDs is shown in **Figure 7A**. L2 and L3 have similar RMSD and RMSF profiles (**Figures 7B,C**), whereas in L4 LAT1 undergoes a less marked conformational change. With decreasing k, LME progresses with a linear path (L2) then takes a segmented track to eventually stop near the putative inner gate as also shown by the analysis of distances from Arg141 (**Figure 7D**). In C21 and C23 the substrate is near the putative inner gate, establishing interactions with Phe262 and Thr345. In C22 and C25, LME is near the outer gate; Thr71, Asn404 and Trp405 contribute significantly to the total interaction energy computed via IFFP. The remaining clusters, instead, are representative of Brownian motions.

The analysis of DIT path along LAT1 transport channel (**Supplementary Figure 5**), shows that the inhibitor neither reaches the intracellular side of the protein nor bypasses the outer gate residue Phe252. In fact, as the spring constant k decreases, DIT progressively fails to reach the deepest regions along the channel: (i) at k = 90 (D2) it is unable to cross the binding site, but bypasses the gate residue Phe252, (ii) at k = 80 (D3) it stops above the outer gate Phe252, and (iii) at k = 70 (D4) it remains in the extracellular domain, never reaching the outer gate. LAT1 rearrangements from OF to IF

FIGURE 6 | Geometrical changes of LAT1 during 5 ns tMD simulations of Tyr. (A) shows the starting position of Tyr in the tMD simulation with LAT1 OF structure into the POPC bilayer membrane. (B) shows RMSD profiles vs. time for LAT1 during OF → IF transitions. RMSD was computed for the α-carbons. (C) shows the RMSF for the three dynamics, computed for α-carbons. α-Helices are represented as red lines. (D) shows distances between Arg141 and substrates molecules. The points mark representative clusters.

RMSF for the three dynamics, computed for α-carbons. α-Helices are represented as red lines. (D) shows distances between Arg141 and LME. The points mark representative clusters.

conformation occurring in D2, D3, D4 tMDs and DIT paths across LAT1 are shown in **Supplementary Movies 8–10**. The starting configuration of DIT in tMDs is shown in **Figure 8A**. The RMSD (**Figure 8B**) and RMSF profiles (**Figure 8C**) confirm the same general behavior previously described for other tMD simulations. In no case DIT passes through the channel, reaching the intracellular side, in agreement with its known inhibitory activity (**Figure 8D**). Varying the spring constant value k, DIT stops near Arg141 for approximately 1 ns (D2); then it remains in a putative outer binding site for approximately 0.5 ns (D3), and finally it stops in the extracellular side, never entering the transport channel of LAT1 for the whole simulated time (D4). From cluster analysis, in D2, a very populated cluster (C28, 93% of population) describes the DIT Brownian motions in the extracellular side. A second cluster, C29, describes the initial transport step, in which DIT enters the binding site, near the outer gate Phe252, and interacts with Ser401 and Lys77. Phe252 is exposed and in an open configuration. C31 shows Brownian motions of DIT in LAT1 extracellular side; C32 is representative of DIT motions at the entrance of the transport channel, where DIT interacts with Trp405 and Ser249. For D4, all clusters

FIGURE 8 | Geometrical changes of LAT1 during 5 ns tMD simulations of DITs. (A) shows the starting position of LME in the tMD simulation with LAT1 OF structure into the POPC bilayer membrane. (B) shows RMSD profiles vs. time for LAT1 during OF → IF transitions. RMSD was computed for the α-carbons. (C) shows the RMSF for three dynamics, computed for the α-carbons. α-helices are represented as red lines. (D) shows distances between Arg141 and DIT. The point mark the representative clusters.

represent DIT Brownian motions in the extracellular side of the membrane.

Taken together, data from D2-4 tMD suggest that DIT inhibits LAT1 transport of amino acids with a competitive mechanism based on a molecular recognition that occurs outside the transport channel. Indeed, DIT may be recognized by LAT1 as a "fake substrate," because of its similarity with the canonical transported substrates, but it can establish interactions only with LAT1 residues above Phe252 and external to the transport channel, preventing the interaction with, and the transport of, other substrates.

### Transport Across LAT1

To summarize evidences collected via our in silico approach (Figure 9.A), it is possible to recognize Phe252 as the key gating residue in LAT1 outer region, as already described (Napolitano et al., 2017a); it is also possible to identify a more extended region around Phe262 that participates in the inner gating mechanism. This region includes also Phe244, Tyr259, Thr345, and Arg348. Phe262 closes the channel by steric hindrance, preventing substrate exit, in synergy with Arg348 (**Figure 9B**).

Ser342, already proposed as a residue involved in LAT1 binding (Geier et al., 2013), is crucial in substrate docking prior to translocation, as demonstrated both in silico and in vitro (Napolitano et al., 2017a).

To further support these findings, there is correspondence between Phe252 in LAT1 and the gating residue Trp202 in AdiC (Geier et al., 2013; Napolitano et al., 2017a). For other transporters of the APC family the transport mechanism is fully characterized: AdiC has a well-defined 2-gates mechanism (Trp202 and Trp293) (Krammer et al., 2016), whereas LeuT has a diffuse gating that involves more amino acids along the transport channel (Zhao and Noskov, 2013). On the other hand, Phe262 and the extended region around it, which could represent a second aromatic gate, suggest that LAT1 transport mechanism may be intermediate between those of AdiC and LeuT. In addition, Tyr tMD simulations show that Tyr259 is the residue contributing the largest energy to substrate interaction; this residue appears to have a role in stabilizing the substrate inside the channel.

Even though no experimental data on an inner gate are currently available for LAT1, our in silico results provide a first plausible description of its transport mechanism at an atomistic level, and can guide further in vitro experiments to confirm the role of the residues involved both in recognition and translocation of the transported substrates or inhibitors.

Finally, we analyzed the paths of water molecules across LAT1 channel through the whole simulation, for every tested condition. Overall, multiple inlet and outlet clusters appear near both channel entrances and the path analysis reveals that only few water molecules cross the entire channel. Instead, most paths link clusters on the same side of the protein and this is probably due to a gating mechanism that prevents the passage across the transport channel.

### CONCLUSIONS

With our research, based on single continuous tMD simulations, we propose, for the first time and at an atomistic level, the characterization of residues involved in LAT1 transport mechanism. The templates selected for modeling the OF and IF LAT1 states are the most suitable so far available, and have been extensively used in recent publications. Thanks to the application of different spring constant (k) values for each tested molecule, we identified a specific computational setup, through which we could investigate the transport mechanism of well-known transported substrates and inhibitor of LAT1. Even though the LAT1 OF → IF transition is forced by tMD simulations, DIT cannot pass through the LAT1 transport channel in specific

### REFERENCES

scenarios which allow Tyr and LME to pass through. Specifically, decreasing k relaxed the transport mechanism, and enabled differential behaviors for transported substrates vs. inhibitor.

With the use of an evolving constraint, tMD does not sample an equilibrium ensemble, therefore not allowing to compute equilibrium properties, such as free energy profiles along the simulated processes. In addition, the presence of a constraint may force the system to overcome very high energy barriers. Despite these limitations, tMD is a very effective and acknowledged approach for investigating transition mechanisms (Ma et al., 2000; van der Vaart and Karplus, 2005; Ovchinnikov and Karplus, 2012). In this perspective, tMD overcomes classical MD methodologies, which are unable to capture large structural rearrangements for an all-atoms system over a suitable time scale. Our tMD simulations can discriminate between transported substrates and inhibitors, and can be proposed for developing novel inhibitors, useful for further pharmacological applications.

### ETHICS STATEMENT

Authors are required to state the ethical considerations of their study in the manuscript, including for cases where the study was exempt from ethical approval procedures. Does the study presented in the manuscript involve human or animal subjects: No.

### AUTHOR CONTRIBUTIONS

LP set up the technique, performed part of the simulations, conducted part of the analyses and drafted the manuscript. CP conducted part of the analyses and revised the manuscript. TL and UG performed part of the simulations and contributed to the data analysis. CI and IE conceived the concept of this work. EG and IE supervised the work and revised the manuscript.

### FUNDING

This research was supported by grants from MIUR Progetto Eccellenza. IE gratefully acknowledges departmental Linea 2 - Azione A 2017 funding.

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fchem. 2018.00350/full#supplementary-material

by an amino acid antiporter. Proc Natl Acad Sci. U.S.A. 108, 3935–3940. doi: 10.1073/pnas.1018081108

acid transporter and its relevance to human health. Front. Chem. 6:243. doi: 10.3389/fchem.2018.00243

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Palazzolo, Parravicini, Laurenzi, Guerrini, Indiveri, Gianazza and Eberini. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Activation of the Calcium-Sensing Receptor Corrects the Impaired Mitochondrial Energy Status Observed in Renal Polycystin-1 Knockdown Cells Modeling Autosomal Dominant Polycystic Kidney Disease

Annarita Di Mise1 , Marianna Ranieri 1 , Mariangela Centrone1 , Maria Venneri 1 , Grazia Tamma1,2, Daniela Valenti 3 \* and Giovanna Valenti 1,2,4 \*

### Edited by:

Graça Soveral, Universidade de Lisboa, Portugal

### Reviewed by:

Kamel Laghmani, INSERM U1138 Centre de Recherche des Cordeliers, France Francesco Moccia, University of Pavia, Italy

\*Correspondence:

Giovanna Valenti<EMAIL_ADDRESS>Daniela Valenti<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Cellular Biochemistry, a section of the journal Frontiers in Molecular Biosciences

> Received: 23 April 2018 Accepted: 03 August 2018 Published: 24 August 2018

#### Citation:

Di Mise A, Ranieri M, Centrone M, Venneri M, Tamma G, Valenti D and Valenti G (2018) Activation of the Calcium-Sensing Receptor Corrects the Impaired Mitochondrial Energy Status Observed in Renal Polycystin-1 Knockdown Cells Modeling Autosomal Dominant Polycystic Kidney Disease. Front. Mol. Biosci. 5:77. doi: 10.3389/fmolb.2018.00077 1 Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Bari, Italy, 2 Istituto Nazionale di Biostrutture e Biosistemi, Rome, Italy, 3 Institute of Biomembranes Bioenergetics and Molecular Biotechnologies, National Council of Research, Bari, Italy, 4 Center of Excellence in Comparative Genomics, University of Bari, Bari, Italy

Autosomal Dominant Polycistic kidney Disease (ADPKD) is a renal channelopathy due to loss-of-function mutations in the PKD1 or PKD2 genes, encoding polycystin-1 (PC1) or polycystin-2 (PC2), respectively. PC1 is a large protein found predominantly on the plasma membrane where interacts with different proteins, including PC2. PC2 is a smaller integral membrane protein also expressed in intracellular organelles, acting as a non-selective cation channel permeable to calcium. Both PC1 and PC2 are also localized to the primary cilium of renal epithelial cells serving as mechanosensor that controls calcium influx through the plasma membrane and regulates intracellular calcium release from the endoplasmic reticulum. The mechanisms by which PC1/2 dysfunction leads to ADPKD needs still to be clarified. We have recently reported that selective Calcium-Sensing Receptor (CaSR) activation in human conditionally immortalized Proximal Tubular Epithelial cells deficient for PC1 (ciPTEC-PC1KD), deriving from urine sediments reduces intracellular cAMP and mTOR activity, and increases intracellular calcium reversing the principal ADPKD dysregulations. Reduced cellular free calcium found in ADPKD can, on the other hand, affect mitochondrial function and ATP production and, interestingly, a relationship between mitochondria and renal polycystic diseases have been suggested. By using ciPTEC-PC1KD as experimental tool modeling of ADPKD, we show here that, compared with wild type cells, ciPTEC-PC1KD have significantly lower mitochondrial calcium levels associated with a severe deficit in mitochondrial ATP production, secondary to a multilevel impairment of oxidative phosphorylation. Notably, selective CaSR activation with the calcimimetic NPS-R568 increases mitochondrial calcium content close to the levels found in resting wild type cells, and fully recovers the cell energy deficit associated to the PC1 channel disruption. Treatment of ciPTEC-PC1KD with 2-APB, an IP3R inhibitor, prevented the

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rescue of bioenergetics deficit induced by CaSR activation supporting a critical role of IP3Rs in driving ER-to-mitochondria Ca2+ shuttle. Together these data indicate that, besides reversing the principal dysregulations considered the most proximal events in ADPKD pathogenesis, selective CaSR activation in PKD1 deficient cells restores altered mitochondrial function that, in ADPKD, is known to facilitate cyst formation. These findings identify CaSR as a potential therapeutic target.

Keywords: calcium-sensing receptor, renal channelopathies, calcimimetics, ciPTEC, mitochondria, ATP

### INTRODUCTION

Loss-of-function of polycystin-1 (PC1) or polycystin-2 (PC2), two trans-membrane proteins that form a heteromeric molecular complex in the cilia and plasma membrane, results in the most common life-threatening genetic renal disorder, called Autosomal Dominant Polycystic Kidney Disease (ADPKD), characterized by the formation and development of kidney cysts (Torres et al., 2007). Biochemical experiments have shown that PC1 and PC2 form a receptor-ion channel complex (Hanaoka et al., 2000; Yu et al., 2009) leading ADPKD to be classified among channelopathies, a heterogeneous group of disorders caused by the dysfunction of ion channels expressed in cellular membranes and many intracellular organelles.

ADPKD affects 1:400–1:1,000 worldwide and 50% of PKD patients will require dialysis or kidney transplantation within 60 years of age (Harris and Torres, 2014). It results from mutations in PKD1 or PKD2 genes (encoding PC1 or PC2, respectively) (Hughes et al., 1995; Mochizuki et al., 1996) which cause analogous clinical signs. The close identity of the disease manifestations, independently of the responsible gene, suggests that PC1 and PC2 are involved in a common signaling pathway.

PC1 is considered a mechanosensor receptor with a large extracellular N-terminal, 11 transmembrane domains and a cytoplasmic C-terminal region (Hughes et al., 1995; Oatley et al., 2012). The N-terminal domain contains several structural motifs found to be essential for interactions with extracellular proteins and carbohydrates (Löhning et al., 1996). The Cterminal cytoplasmic domain contains a coiled-coil sequence that binds specifically to the C-terminus of PC2 (Qian et al., 1997), and it has been shown that, once cleaved from the full length PC1, it translocates to the nucleus (Chauvet et al., 2004; Chapin et al., 2010). Interestingly, PC2 expression is able to attenuate PC1 C-terminal tail translocation, modulating its signaling properties (Chauvet et al., 2004).

PC2, also known as transient receptor potential cation channel, subfamily P, member 2 (TRPP2), is a nonselective cation channel containing 6 transmembrane domains, with both Nand C-terminal tails facing the cytoplasm (Mochizuki et al., 1996; Celic´ et al., 2008). The N-terminal portion carries a ciliary targeting domain (Geng et al., 2006), while the C-terminal tail includes an EF-hand, and a coiled coil motif whose binding to the coiled-coil domain in the PC1 C terminus is critical for the formation of the PC1-PC2 complex (Yu et al., 2009). Conversely, other experiments showed that PC1 and PC2 interaction is preserved in systems not expressing the coiled-coil domain and is dependent on the N-terminal domain (Babich et al., 2004; Feng et al., 2008; Celic et al., 2012 ´ ). However, the factors that regulate the localization, trafficking, and channel activity of the polycystins remain still unclear.

The polycystins form a complex that localizes to the primary cilium, where it may be involved in chemosensory or mechanosensory pathways (Pazour and Rosenbaum, 2002; Chapin and Caplan, 2010). In addition, the PC complex is expressed on the plasma membrane, as well as in intracellular compartments, where PC2 can regulate Ca2+ release from the endoplasmic reticulum (Koulen et al., 2002). Many studies propose that PC1 and PC2 may reciprocally affect each other's membrane or ciliary localization (Harris et al., 1995; Ong and Wheatley, 2003; Xu et al., 2007), and importantly, the interaction between the polycystins has been suggested to be crucial in defining the properties of the ion channel associated with the complex (Hanaoka et al., 2000; Delmas et al., 2004).

In ADPKD, disruption of the PC complex, resulting from the loss of function of PKD1 or PKD2, cause the dysregulation of many pathways such as cAMP, calcium and mTOR signaling cascades, which promote cell proliferation and apoptosis. The hallmark of the disease is the spontaneous generation and constant growth of kidney cysts which gradually expand to demolish the normal renal parenchyma, leading to end stage renal disease (ESRD).

We have recently shown that selective activation of the extracellular calcium-sensing receptor (CaSR) in human conditionally immortalized proximal tubular epithelial cells (ciPTEC), silenced for PKD1 or generated from an ADPKD1 patient, increases cytosolic calcium, and reduces intracellular cAMP and mTOR activity, reversing the principal dysregulations considered the most proximal events in ADPKD pathogenesis, making CaSR a possible candidate as therapeutic target (Di Mise et al., 2018). On the other hand, reduced intracellular calcium observed in ADPKD can affect mitochondrial function and ATP production with consequence on glucose metabolism (Rowe et al., 2013), as showed in recent studies which reported defective glucose metabolism in ADPKD-affected cells and tissues (Rowe et al., 2013; Menezes et al., 2016). They demonstrated that PC1 lacking cells consume high levels of glucose, preferentially using it in aerobic glycolysis for their energy production (Rowe et al., 2013). Indeed, mitochondrial abnormality exists from an early phase of ADPKD, underlining a relationship between mitochondria and renal polycystic diseases (Li et al., 2012; Rowe et al., 2013; Magistroni and Boletta, 2017; Padovano et al., 2017). In line, an association between mitochondria abnormalities and cystogenesis has been reported in cyst-lining cells in ADPKD model mice and in rats (Ishimoto et al., 2017). Recent clinical studies indicate that oxidative stress is already present in the early stages of ADPKD, even when renal function is preserved (Menon et al., 2011; Klawitter et al., 2014), showing that mitochondria, representing the primary reactive oxygen species source, play a functional role in cyst formation. However, the precise pathophysiological role of mitochondria in ADPKD remains elusive.

We now report that CaSR activation in ciPTEC stably knocked down for PC1 restores the decreased mitochondrial calcium levels and fully reverse the deficient ATP production.

### MATERIALS AND METHODS

### Materials

All chemicals were purchased from Sigma (Sigma-Aldrich, Milan, Italy). NPS-R568 was kindly gifted by Amgen (Amgen Dompé S.p.a., Milan, Italy). Media for cell culture were from Lonza (Lonza s.r.l., Milan, Italy). pcDNA-4mtD3cpv was a gift from Amy Palmer & Roger Tsien (Addgene plasmid #36324).

### Antibodies

Monoclonal CaSR antibody recognizing amino-acid 15–29 at the extracellular N-terminus was from Sigma-Aldrich, Milan, Italy. Secondary goat anti-mouse IgG biotin antibodies were purchased from Sigma-Aldrich, Milan, Italy. Streptavidin-488 conjugate were from Alexa Fluor (Molecular Probes, Eugene, Oregon, USA).

### Generation of ciPTEC Knocked Down for Polycystin-1

ciPTEC were generated as described by Wilmer et al. (2010). Briefly, primary cells were cultured by collecting mid-stream urine within 5 h after collection. Urine sediment was resuspended in DMEM Ham's F12 medium supplemented with 10% fetal bovine serum (FBS), 100 IU/ml penicillin, 100µg/ml streptomycin, ITS (5µg/ml insulin, 5µg/ml transferrin and 5 ng/ml selenium), 36 ng/ml hydrocortisone, 10 ng/ml epidermal growth factor (EGF), and 40 pg/ml triiodothyronine. The suspension was placed at 37◦C in a 5% CO2 incubator.

Primary cells were immortalized as previously described (Wilmer et al., 2010). Cells were infected with SV40T and hTERT vectors, containing, respectively, geneticin (G418) and hygromycin resistance (O'hare et al., 2001; Satchell et al., 2006). Subconfluent cell layers were then grown at 33◦C and selected by using G418 (400µg/ml) and hygromycin B (25µg/ml) for 10 days. Stable knocked down ciPTEC for polycystin-1 (ciPTEC-PC1KD) were obtained transducing a cloned ciPTEC line (ciPTECwt) of a healthy individual (34.8) by adding lentiviral vectors encoding miR-shRNA directed against polycystin-1, cloned in tandem (pCHMWS Bsd 2xmiRNA PKD1), to the culture medium (Mekahli et al., 2012). Transduced cells were selected using 10 g/ml blasticidin. Experiments were performed prior cellular maturation for 11 days at 37◦C. The reduced expression of PC1 was showed by Mekahli and coworkers which biochemically characterized these cell lines (Mekahli et al., 2012).

### Measurement of Mitochondrial ATP Production Rate in ciPTEC

The rate of mitochondrial ATP production was determined in ciPTEC permeabilized with digitonin (0.01%) essentially as already reported (Valenti et al., 2016). Briefly, cells (0.3– 0.4 mg protein) were incubated at 37◦C in 2 ml of respiratory medium consisting of 210 mM mannitol, 70 mM sucrose, 3 mM MgCl2, 20 mM Tris/HCl, 5 mM KH2PO4/K2HPO4, (pH 7.4) plus 5 mg/ml BSA, in the presence of the coupled enzyme system revealing ATP, indicated as ATP detecting system (ATPds), containing the substrates glucose (2.5 mM) and NADP+ (0.25 mM) and the coupled enzymes hexokinase (HK, 3 e.u.) and glucose 6-phosphate dehydrogenase (G6P-DH, 2 e.u.). The assay has been performed in the presence of 0.01 mM diadenosine pentaphosphate (Ap5A) to selectively inhibit the mitochondrial adenylate kinase. The measure has been carried out by adding as energy sources, either glutamate (GLU) plus malate (MAL) (5 mM each) or succinate (SUCC, 5 mM) plus rotenone (ROT, 3µM), or ascorbate (ASC, 0.5 mM) plus N,N,N′ ,N′ - tetramethyl-p-phenylenediamine (TMPD, 0.25 mM). After 5 min of incubation with digitonin (0.01% w/v), ADP (0.5 mM) was added to start the reaction, and the reduction of NADP+ in the extra-cellular phase was monitored as an increase in absorbance at 340 nm. As a control, the ATP synthase inhibitor oligomycin (OLIGO, 5 µg/10 µl) was added during the reaction to ascertain the inhibition of the mitochondrial ATP production. Attention was reserved to use excess HK/G6P-DH coupled enzymes to guarantee a non-limiting ADP-restoring system for the assay of ATP production.

### Measurement of ATP Levels in ciPTEC

ciPTEC were detached from plate, washed with PBS and cellular ATP was extracted by using the boiling water procedure, as described in Yang et al. (2002). The amount of intracellular ATP was determined enzymatically in the extracts, as described in Valenti et al. (2010). Cells were left under basal condition or stimulated with NPS-R568 (10µM for 60 min at 37◦C) in Ringer's solution containing 120 mM NaCl, 4 mM KCl, 15 mM NaHCO3, 1 mM MgCl2, 15 mM Hepes, 0.5 mM NaH2PO4, 10 mM Glucose, 2 mM CaCl2, 0.5 mM Na2HPO4, 0.4 mM MgSO4, pH 7.4 (modified by Mekahli et al., 2012; Miyakawa et al., 2013).

### Fluorescence Resonance Energy Transfer (FRET) Measurements

To evaluate mitochondrial calcium content, fluorescence resonance energy transfer (FRET) experiments were performed as described (Di Mise et al., 2015, 2018). Briefly, ciPTEC were seeded onto 20-mm glass coverslips at 37◦C for 11 days and were transiently transfected with a plasmid encoding a mitochondrially targeted cameleon containing a mutant calmodulin (mCaM) sequence cloned between CFP and circularly permuted Venus (cpV) (Palmer et al., 2006). Experiments were performed 24 h post-transfection. Cells were left under basal condition or stimulated with NPS-R568 (10µM for 60 min at 37◦C) in Ringer's solution, described above, containing 2 mM CaCl2.

FRET measurements were carried out using MetaMorph software (Molecular Devices, MDS Analytical Technologies, Toronto, Canada). CFP and YFP were excited at 436 and 500 nm, respectively; fluorescence emitted was measured at 480/40 nm for CFP and 535/30 nm for YFP and FRET. Corrected normalized FRET values were determined as already described (Rodighiero et al., 2008; Tamma et al., 2013, 2017; Russo et al., 2017). Each image was corrected for CFP cross-talk and YFP cross-excitation. Therefore, netFRET = [IFRETbg – ICFPbg·K1 – IYFPbg·(K2 αK1)]/(1-δK1) where IFRETbg, ICFPbg, and IYFPbg are the background-corrected pixel gray values measured in the FRET, CFP ,and YFP windows, respectively; K1, K2, α, and δ are calculated to evaluate the crosstalk between donor and acceptor. The integrated fluorescence density values of the images from each cell were analyzed using MetaMorph and Microsoft Excel software.

### Immunofluorescence Microscopy

CaSR immunofluorescence localization in polarized ciPTEC was performed as previously described (Jansen et al., 2014; Di Mise et al., 2015). ciPTEC were cultured on polyester Transwell inserts, left 11 days at 37◦C for maturation, and then fixed using 2% (w/v) paraformaldehyde in HBSS with addition of 2% (w/v) sucrose for 5 min and permeabilized in 0.3% (v/v) triton X-100 in HBSS for 10 min.

Cells were incubated with antibodies diluted in block solution containing 2% (w/v) bovine serum albumin (BSA) and 0.1% (v/v) tween-20 in HBSS against the calcium-sensing receptor (CaSR) at 4 ◦C overnight. Following treatment with secondary rabbit-antimouse-biotin antibodies followed by Streptavidin-488, samples were mounted on glass slides with Mowiol. Images were obtained with a confocal microscope Leica TCS SP2 (Leica Microsystems, Heerbrugg, Switzerland).

### Statistical Analysis

Statistical analysis was performed using One-way ANOVA followed by Newman–Keuls multiple comparisons test or Twoway ANOVA followed by Turkey's multiple comparisons test or t-test. All values are expressed as means ± SEM. A difference of P < 0.05 was considered statistically significant.

### RESULTS AND DISCUSSION

### Impaired Bioenergetics Status in ciPTEC Silenced for PC1

Emerging evidence of inherent metabolic reprogramming in PKD1 knockout cells suggests that the PC1-PC2 complex regulates cellular metabolism (Menezes et al., 2016; Padovano et al., 2017). The exact nature of the metabolic alterations remains controversial, with some groups reporting enhanced glycolysis, reminiscent of the Warburg phenomenon (Rowe et al., 2013; Padovano et al., 2017), and others reporting no evidence for a glycolytic switch (Menezes et al., 2016; Warner et al., 2016), proposing instead fatty acid oxidation impairment (Menezes et al., 2016; Hajarnis et al., 2017).

Lin and collaborators (Lin et al., 2018) suggest a direct link between PKD proteins and control of mitochondrial activity, showing that PKD1 knockout cells have different metabolic fluxes with likely altered oxidoreductase activity, consistent with changes in NAD+/NADH ratio. Furthermore, their data document altered mitochondrial membrane potential and abnormal mitochondrial networks in PKD1 knockout cell lines. Morphological abnormalities of mitochondria have also been found in human ADPKD cyst-derived cells with heterozygous and homozygous PKD1 mutation, indicated to facilitate cyst formation in ADPKD (Ishimoto et al., 2017).

On this basis, to investigate whether the bioenergetics status is affected in ciPTEC stably silenced for PC1, we first evaluated the mitochondrial ATP synthesis in ciPTEC-PC1KD cells compared with wild type clone, by monitoring in continuous the ATP produced by mitochondria in situ and flowed outside cells, under conditions where oxidative phosphorylation (OXPHOS) can take place (Valenti et al., 2010). ciPTEC-PC1KD or ciPTECwt, permeabilized with digitonin (0.01%), were incubated with the ATP detecting system (ATP-ds) and the efflux of ATP in the extracellular phase occurring after the addition of ADP was monitored as an increase in absorbance at 340 nm due to NADPH synthesis, and evaluated as a measure of mitochondrial ATP production (**Figure 1A**).

To give a more complete evidence on the efficiency of the OXPHOS system, and disclose the distinctive contribution of each mitochondrial respiratory chain complex composing the OXPHOS apparatus, the mitochondrial ATP synthesis was measured by supplying as energy sources the respiratory substrates of complex I glutamate plus malate (GLU+MAL), complex II succinate (SUCC) or complex IV ascorbate plus TMPD (ASC+TMPD). As shown by the representative spectrophotometric traces (**Figures 1B–D**) and by the histograms reporting the statistical analyses of data (**Figure 2A**), a drastic reduction in the rate of mitochondrial ATP synthesis was found, commonly shared when supplied ciPTEC-PC1KD with GLU+MAL (estimated as 41 ± 13% vs. ciPTECwt, P < 0.01) or SUCC (estimated as 39 ± 10% vs. ciPTECwt, P < 0.01) or ASC+TMPD (estimated as 56 ± 11% vs. ciPTECwt, P < 0.01), thus indicating a multilevel inhibition of the mitochondrial ATP production by OXPHOS in ciPTEC-PC1KD.

To assess whether the compromised mitochondrial energy efficiency found in ciPTEC-PC1KD could impact the cell energy status, the cellular ATP pool was measured (**Figure 2B**). ATP level was found significantly reduced in ciPTEC-PC1KD (estimated as 38 ± 4%, P < 0.0001) with respect to wt, suggesting that the deficit in ATP production by mitochondria reflects and mirror the overall cellular energy level in ciPTEC-PC1KD.

These data indicate an impairment in mitochondrial OXPHOS associated with a great shortage in cell energy status in PKD1 silenced cells. How the lack of PKD1 causes such a mitochondrial dysfunction in ciPTEC it is not yet clear. Both transcriptional and/or post-translational mechanisms regulating OXPHOS process may be involved. Among post-translational regulation of OXPHOS, calcium signaling between cytosol and matrix primarily coordinates the crosstalk of mitochondria within the cell, establishing the balance between the energy requests of the cell and the energy production by mitochondrial OXPHOS (Glancy and Balaban, 2012).

FIGURE 1 | Impaired mitochondrial ATP synthesis in human ciPTEC-PC1KD. (A) Schematic representation of the mitochondrial ATP synthesis revealed by ATP detecting system (ATP-ds). ANT, adenine nucleotide translocator; AK, adenylate kinase; HK, hexokinase; G6P-DH, glucose 6-phosphate dehydrogenase; 6P-GLUCδ-LAC, 6 phosphogluconate-δ-lactone; IMS, intermembrane space; MIM, mitochondrial inner membrane; OM, outer membrane. (B–D) Representative spectrophotometric traces of mitochondrial ATP production in human ciPTEC. Either human ciPTEC stably silenced for polycystin-1 (ciPTEC-PC1KD) or wild type clone (ciPTECwt) (0.3 mg) were permeabilized with 0.01% digitonin for 5 min and incubated at 37◦C in 2 ml of respiration medium in the presence of ATP-ds plus the indicated respiratory substrates. (B) Glutamate plus malate (GLU/MAL, 5 mM each), (C) 5 mM succinate (SUCC) plus 3µM rotenone, (D) 5 mM ascorbate plus 0.5 mM TMPD (ASC/TMPD). Where indicated, ADP (0.5 mM) was added. At the arrows, the ATP synthase inhibitor oligomycin (OLIGO, 5 µg/10 µl) was added in course of reaction. The numbers along curves represent the rates of the increase in absorbance at 340 nm, measured as tangents to the initial slopes and expressed as nmol of NADPH formed/min per mg of protein.

FIGURE 2 | Impaired bioenergetics status in human ciPTEC-PC1KD. (A) The rate of mitochondrial ATP production was measured in human ciPTEC stably silenced for polycystin-1 (ciPTEC-PC1KD) vs. wild type clone (ciPTECwt), in the presence of the respiratory substrates GLU plus MAL (GLU/MAL, 5 mM each) or 5 mM SUCC plus 3µM rotenone, or 5 mM ASC plus 0.5 mM TMPD (ASC/TMPD), as described in the legend of Figure 1. Values are means ± SEM (\*P < 0.01 vs. ciPTECwt) obtained from three independent experiments and expressed as nmol NADPH/min per mg protein. (B) Cellular ATP levels were measured in ciPTEC-PC1KD vs. matched ciPTECwt, as described under Methods. Each histogram is representative of four independent experiments. Data are expressed as means ± SEM. Significant differences with respect to wt were calculated by Student's t-test (\*\*\*\*P < 0.0001).

### Reduced Mitochondrial Calcium Content in ciPTEC-PC1KD

It has been shown that cultured epithelial cells derived from human ADPKD cysts exhibit a reduction in steady state cytosolic calcium levels with respect to normal human kidney cells (Yamaguchi et al., 2006). Moreover, PC complex interacts with other calcium channels expressed in the ER, displaying a pivotal role in the prevention of intracellular stores depletion, especially of the ER itself (Anyatonwu et al., 2007; Weber et al., 2008; Santoso et al., 2011). Recently, we demonstrated that, compared to ciPTECwt, ciPTEC-PC1KD have significantly lower calcium concentration both in the cytosol and in the ER, highlighting that the loss-of-function mutation in PKD1 is strictly connected to the dysregulation of the two intracellular calcium bulks, likely secondary to the PC complex disruption and dysfunction (Di Mise et al., 2018).

The ER is known to be functionally associated to mitochondria with a rapid transport of calcium across their membranes and its accumulation in the mitochondrial matrix, where several calcium effectors are located (Tarasov et al., 2012). The inositol 1,4,5-trisphosphate receptors (IP3Rs) are the main drivers of the ER-to-mitochondria Ca2+ shuttle supporting cellular bioenergetics (Cárdenas et al., 2010). Mitochondrial calcium uptake by IP3R-released Ca2+ is fundamentally required to maintain adequate mitochondrial NADH production to sustain OXPHOS in resting cells (Cárdenas et al., 2010). IP3R activity is linked to regulation of cellular bioenergetics through calcium-dependent activation of mitochondrial ATP synthesis, by triggering key matrix enzymes, including pyruvate dehydrogenase, α-oxoglutarate dehydrogenase, isocitrate dehydrogenase, as well as downstream elements of OXPHOS, comprising F1/Fo ATP synthase and the cytochrome chain (Glancy and Balaban, 2012).

In line with these findings, the resting mitochondrial calcium content in ciPTEC-PC1KD resulted significantly reduced with respect to wt cells (ciPTEC-PC1KD = 88.05 ± 2.6%, n = 140, vs. ciPTECwt = 100%, n = 192; P < 0.01; **Figure 3**). These data further confirm the functional interaction between ER and mitochondria that are in close apposition, allowing calcium transfer between these organelles and supporting the hypothesis that the PCs are involved in the regulation of mitochondrial calcium levels, as previously suggested (Patergnani et al., 2011; Padovano et al., 2017). Indeed, in line with this hypothesis, PCs have been shown to co-fractionate with mitochondria-associated ER membranes (MAMs) (Padovano et al., 2017).

### Activation of CaSR by NPS-R568 Results in Full Recovery of Mitochondrial Calcium Levels and Restoring of Cell Energy Deficit in ciPTEC-PC1KD

ciPTEC stably silenced for PC1 as well as wild type clone, express endogenous CaSR mainly localized to the apical plasma membrane as it occurs in native renal proximal tubule epithelial cells (**Figure 4**) (Riccardi and Valenti, 2016; Di Mise et al., 2018). CaSR is coupled to three main groups of heterotrimeric G proteins, Gq/11, Gi, and G12/13. Recently we have shown that

CaSR expressed in ciPTEC interacts with Gq as a downstream effector (Di Mise et al., 2015). As known, when CaSR is activated, it induces cytosolic calcium increase via Gq activation which stimulates PLC with subsequent IP3 and diacylglycerol production. IP3 binds to high affinity receptors on the ER (IP3Rs), causing the release of calcium into the cytoplasm.

#P < 0.001 ciPTECwt+NPS-R568 vs. ciPTECwt Basal).

On this basis, we evaluated the effect of CaSR stimulation with NPS-R568 on mitochondrial calcium levels. NPS-R568 treatment resulted in a significant increase in ciPTEC-PC1KD mitochondrial calcium (ciPTEC-PC1KD+NPS-R568 = 97.17 ± 2.87%, n = 200), which raised close to the basal levels observed in ciPTECwt (**Figure 3**). This result suggests that the increase in mitochondrial calcium is a consequence of the CaSR activation induced signaling which leads to IP3 production and calcium influx from ER to the mitochondrion through IP3Rs (Cárdenas et al., 2010). Moreover, the increase of cytosolic Ca2+ elicited by CaSR activation also contributes to the increment of mitochondrial Ca2+ content via the mitochondrial calcium uniporter (MCU) (Marchi and Pinton, 2014).

As known, mitochondrial calcium homeostasis and uptake has a pivotal role in the regulation of mitochondrial ATP generation as well as cytosolic NAD+/NADH metabolism, thus sustaining the energy requirements of the cell (Raffaello et al., 2016; Arduino and Perocchi, 2018).

Remarkably, ciPTEC treatment with the calcimimetic NPS-R568 resulted in a full recovery of cellular ATP content in ciPTEC-PC1KD (ciPTEC-PC1KD+NPS-R568 = 144.3 ± 1.67 nmol/mg protein), restoring the levels measured in ciPTECwt at basal conditions (ciPTECwt = 139.2 ± 2.27 nmol/mg protein; **Figure 5A**). To assess the strict involvement of calcium transfer from ER to mitochondria in sustaining cellular bioenergetics thus rescuing energy deficit in PC1 knockdown cells after CaSR activation, ciPTEC were treated with 2-aminoethoxydiphenyl borate (2-APB), a selective IP3R

membrane localization as occurs in native proximal tubule epithelial cells.

inhibitor (Szatkowski et al., 2010). Furthermore, to abolish the calcium amount permeating mitochondria directly from the cytosol via MCU, we blocked IP3Rs with 2-APB in presence of BAPTA-AM, a cell-permeant highly selective calcium chelator (Gerbino et al., 2009). 2-APB treatment of ciPTEC-PC1KD completely prevented the rescue of bioenergetics deficit induced by NPS-R568, showing values comparable to the intracellular ATP content measured under basal conditions (70.6 ± 0.9 nmol/mg protein; **Figure 5B**). A similar reduction in cellular ATP was reported in ciPTECwt treated with 2-APB (82.4 ± 0.82

FIGURE 6 | Models of intracellular calcium signaling network and mitochondrial calcium regulation of cellular bioenergetics in wt, PC1KD, and calcimimetic-treated PC1KD cells. Mitochondria are main players in calcium signaling network, skillful in regulating both the extent and the spatial/temporal Ca2+ signals. The membrane micro-domains between the ER and mitochondria -named Mitochondria Associated Membranes (MAMs)- are critical for an efficacy inter-organelle crosstalk (Rieusset, 2018). Several proteins, including IP3R, grp75, and VDAC, are involved in Ca2+ release from the ER, ensuring an active mitochondrial Ca2+ uptake. The mitochondrial Ca2+ uniporter (MCU) and the H+/Ca2+ exchanger (LETM1) mediate the Ca2+ uptake from cytosol inside mitochondria; conversely, the mitochondrial Na+/Ca2+ exchanger (NCLX), catalyzes the mitochondrial Ca2+ export. Inside the mitochondrion are schematized the interactions of matrix Ca2+ with processes involved in oxidative phosphorylation. The red arrows from Ca2+ to the different protein targets indicate either a direct or indirect modulating effect on their enzymatic or transport activities. Under basal conditions, PC1KD cells show lower mitochondrial Ca2+ levels with respect to wt, associated to a multilevel inhibition of OXPHOS apparatus, leading to a severe deficit of mitochondrial ATP production and cellular energy status. CaSR stimulation with the calcimimetic NPS-R568 rises intracellular calcium levels triggering the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) which drives Ca2+ from the cytosol to the lumen of the ER. CaSR activation prompts phospholipase C (PLC), a phosphodiesterase responsible for the hydrolysis of phosphatidylinositol 4,5-biphosphate to acil-glycerol and inositol 3 phosphate (IP3). The binding of IP3 to its receptor IP3R induces Ca2+ release from ER and transfer of Ca2+ from ER to mitochondria at the contact sites between ER and mitochondria. CaSR activation by calcimimetic increases mitochondrial Ca2+ levels and full restores the bioenergetics deficit in PC1KD. Enzyme, channel, and carrier abbreviations: PDH, pyruvate dehydrogenase; CS, citrate synthase; ACase, aconitase; IDH, isocitrate dehydrogenase; OGDH, oxoglutarate dehydrogenase; SCS, succinyl CoA synthase; SDH, succinate dehydrogenase (component of Complex II); Fase, fumarase; MDH, malate dehydrogenase; VDAC, voltage-dependent anion channel; ANT, adenine nucleotide translocator; IP3R, inositol 3-phosphate receptor. The complexes of oxidative phosphorylation are labeled as roman numerals from I to IV.

nmol/mg protein; **Figure 5B**), suggesting a critical role of IP3Rs in driving ER-to-mitochondria Ca2+ shuttle supporting cellular bioenergetics. BAPTA-AM per se did not affect ATP cellular levels (data not shown).

These findings point to a crucial role of mitochondrial calcium in regulating the mitochondrial energy status. Indeed, calcium is actively transported inside mitochondria and accumulates in the mitochondrial matrix, where several calcium protein targets are located (Rizzuto et al., 2012) (details in the model in **Figure 6**). In this context, the existence of high-calcium micro-domains between ER and mitochondria ensures efficient calcium transfer from ER to mitochondria (Rizzuto et al., 1998; Csordás et al., 1999, 2010), which is mediated by a multiprotein complex composed—among others—of the IP3R at ER membrane and of the VDAC at outer mitochondrial membrane (OMM) (Szabadkai et al., 2006). The zones of close contact between the ER and mitochondria, called Mitochondria Associated Membranes (MAMs), are crucial for a correct crosstalk between the two organelles (Csordás et al., 2006). Therefore, MAMs disruption causes the suppression of the IP3-mediated release of calcium from the ER to mitochondria, with a consequent reduction of ATP production (Rowland and Voeltz, 2012).

A model of intracellular calcium signaling network in PC1 deficient cells exposed to calcimimetic is shown in **Figure 6**.

To summarize, the present contribution provides the first evidence that selective CaSR activation in human ciPTEC stably knocked down for PC1, restores mitochondrial calcium content and fully rescues the bioenergetics dysfunction known to facilitate cysts formation in ADPKD. These data, together with our previous demonstration that CaSR activation in ciPTEC-PC1KD increases cytosolic calcium and decreases cAMP and mTOR activity, indicate that CaSR signaling can reverse the principal dysregulations considered the most proximal events

### REFERENCES

in ADPKD pathogenesis, making CaSR a potential candidate as therapeutic target.

### AUTHOR CONTRIBUTIONS

GV and DV designed research and supervised the project. DV and AD designed, performed, and interpreted experiments. MR, MC, MV, and GT performed research and analyzed data. AD, GV, and DV wrote the paper. All authors commented on the manuscript.

### FUNDING

This study was supported in part by Telethon funding (grant number GGP13227) and by ASI (Italian Space Agency, grant number 2013-091-R.0).

### ACKNOWLEDGMENTS

We thank Dr. Elena Levtchenko for providing ciPTECwt and ciPTEC-PC1KD cell lines. The authors thank Amgen (Amgen Dompé S.p.a., Milan, Italy) for providing NPS-R568.

line expressing functional influx and efflux transporters. Cell Tissue Res. 339, 449–457. doi: 10.1007/s00441-009-0882-y

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Di Mise, Ranieri, Centrone, Venneri, Tamma, Valenti and Valenti. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Relevance of Copper and Organic Cation Transporters in the Activity and Transport Mechanisms of an Anticancer Cyclometallated Gold(III) Compound in Comparison to Cisplatin

#### Sarah Spreckelmeyer 1,2, Margot van der Zee1 , Benoît Bertrand1,3, Ewen Bodio3 , Stefan Stürup4 and Angela Casini 1,5 \*

1 Department Pharmacokinetics, Toxicology and Targeting, Groningen Research Institute of Pharmacy, University of Groningen, Groningen, Netherlands, 2 Medicinal Inorganic Chemistry Group, Department of Chemistry, University of British Columbia, Vancouver, BC, Canada, 3 ICMUB UMR6302, CNRS, Université Bourgogne Franche-Comté, Dijon, France, 4 Department of Pharmacy, University of Copenhagen, Copenhagen, Denmark, 5 School of Chemistry, Cardiff University, Cardiff, United Kingdom

### Edited by:

Cesare Indiveri, University of Calabria, Italy

#### Reviewed by:

Sara Eyal, Hebrew University of Jerusalem, Israel Francisco Solano, Universidad de Murcia, Spain

> \*Correspondence: Angela Casini<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Chemical Biology, a section of the journal Frontiers in Chemistry

Received: 29 March 2018 Accepted: 03 August 2018 Published: 04 September 2018

#### Citation: