**THE TRUTH IN COMPLEXES: WHY UNRAVELING ION CHANNEL MULTI-PROTEIN SIGNALING NEXUSES IS CRITICAL FOR UNDERSTANDING THE FUNCTION OF THE NERVOUS SYSTEM**

**Topic Editors Leigh A. Swayne, Christophe Altier and Gerald W. Zamponi**

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## **THE TRUTH IN COMPLEXES: WHY UNRAVELING ION CHANNEL MULTI-PROTEIN SIGNALING NEXUSES IS CRITICAL FOR UNDERSTANDING THE FUNCTION OF THE NERVOUS SYSTEM**

Topic Editors:

**Leigh A. Swayne,** University of Victoria, Canada **Christophe Altier,** University of Calgary, Canada **Gerald W. Zamponi,** University of Calgary, Canada

Artistic rendering of an ion channel signaling nexus.

In the search for simple explanations of the natural world, its complicated textures are often filed down to a smoothened surface of our liking. The impetus for this Research Topic was borne out of a need to re-ignite interest in the complex – in this case in the context of ion channels in the nervous system. Ion channels are the large proteins that form regulated pores in the membranes of cells and, in the brain, are essential for the transfer, processing and storage of information. These pores full of twists and turns themselves are not just barren bridges into cells. More and more we are beginning to understand that ion channels are like bustling medieval bridges (packed with apartments and shops) rather than the more sleek modern variety – they are dynamic hubs connected with many structures facilitating associated activities. Our understanding of these networks continues to expand as our investigative tools advance. Together these

articles highlight how the complexity of ion channel signaling nexuses is critical to the proper functioning of the nervous system.

# Table of Contents

## *135 The Functionalized Amino Acid (S)-Lacosamide Subverts CRMP2-Mediated Tubulin Polymerization to Prevent Constitutive and Activity-Dependent Increase in Neurite Outgrowth*

Sarah M. Wilson, Aubin Moutal, Ohannes K. Melemedjian, Yuying Wang, Weina Ju, Liberty François-Moutal, May Khanna and Rajesh Khanna

## *150 Novel Endogenous N-Acyl Amides Activate TRPV1-4 Receptors, BV-2 Microglia, and are Regulated in Brain in an Acute Model of Inflammation*

Siham Raboune, Jordyn M. Stuart, Emma Leishman, Sara M. Takacs, Brandon Rhodes, Arjun Basnet, Evan Jameyfield, Douglas McHugh, Theodore Widlanski and Heather B. Bradshaw

## The truth in complexes: perspectives on ion channel signaling nexuses in the nervous system

## *Leigh A. Swayne1,2,3\*, Christophe Altier 4 and Gerald W. Zamponi 4*

*1 Island Medical Program, Division of Medical Sciences, University of Victoria, Victoria, BC, Canada*

*3 Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, BC, Canada*

*4 Department of Physiology and Pharmacology, University of Calgary, Calgary, AB, Canada*

*\*Correspondence<EMAIL_ADDRESS>
#### *Edited and reviewed by:*

*Egidio D'Angelo, University of Pavia, Italy*

**Keywords: interactome, ion channels, signaling networks, protein-protein interactions, protein-lipid interactions**

## **ION CHANNELS AS SIGNALING NEXUSES**

Ion channels are complex hetero-oligomeric structures characterized by large, dynamic interaction networks, or "interactomes." In addition to directing channel localization, density and ion fluxes, these complexes facilitate downstream signaling events. Moreover, pathological modulation of these networks contributes to neurological dysfunction. Our contributors to this Research Topic, *"The truth in complexes: why unraveling ion channel multiprotein signaling nexuses is critical for understanding the function of the nervous system"* have considered interactomes from the perspective of the ion channel, from that of its intracellular protein modulators, and even from the point of view of lipid modulators. Together these diverse perspectives spin an intricate web of ion channel regulation in the nervous system.

## **MAJOR HUB: THE N-methyl-D-ASPARTATE RECEPTOR (NMDAR)**

Described by Fan et al. (2014) as a "multifunctional machine," the NMDAR interacts with a staggering number of proteins to shape synaptic plasticity, psychiatric disorders and ischemic neuronal damage. Notably, the authors outline arguably the most exciting example of interactome-based basic science leading to improved health outcomes: Tat-NR2B9c (also called NA-1). This cellpermeable peptide targets a specific NMDAR interaction, reducing ischemic brain damage in rodents, primates and humans (Sun et al., 2008; Cook et al., 2012; Hill et al., 2012). Li et al. (2014) similarly highlights interactions between several ligandgated channels, including the NMDAR with other receptors and intracellular proteins, again focusing on these interactions as potential therapeutic targets for neuroprotection.

## **NOVEL NODES**

Several other contributions shed light on the new insights into the function and composition of interactomes of various voltagegated channels, regulated leak channels, and so called large pore channels.

#### **VOLTAGE-GATED CHANNELS**

Traditionally viewed as auxiliary subunits, K+ channel regulatory proteins are growing in complexity in terms of function and type. Known to regulate activation and trafficking of muscarinic receptor-activated Kir3 channels, Zylbergold et al. (2014) provide evidence for an additional role of Gβγ subunits in Kir3 channel stability. Nagi and Pineyro (2014) focus specifically on opioid receptor signaling in the regulation of these channels. Jerng and Pfaffinger (2014) describe regulation of another K+ current, sub-threshold A-type (Kv4), by the so-called auxiliary subunits, dipeptidyl peptidase-like proteins (DPLPs) and Kv4 channel interacting proteins (KChIPs). While these were amongst the first identified interactors (e.g., for KChiP An et al., 2000), subsequent studies have significantly expanded the network. With respect to DPLPs and KChIPs, further study has also shed new light on their molecular diversity via alternative splicing as well as their roles in regulating several other channel types, such as voltage-gated Ca2+ channels and NMDARs. Connecting K+ channels with voltage-gated Ca2+ channels, Engbers et al. (2013) review how channel-channel interactions between intermediate conductance Ca2+-activated K+ channels (IKCa) and low voltage-activated Ca2+ channels (Cav3) functionally interact with other conductances to regulate signal processing in the cerebellum.

#### **Na+ LEAK CHANNEL, NALCN**

Elusive until recently, understanding of this regulated leak channel whose loss in mice is lethal (Lu et al., 2007), has greatly expanded by virtue of key insights into its interactome. Cochet-Bissuel et al. (2014) detail its ever-expanding list of interacting proteins, such as the M3 muscarinic receptor (Swayne et al., 2009). The authors highlight the involvement of the NALCN interactome in a number of disorders in the nervous system ranging from autism spectrum disorder (ASD) and schizophrenia to epilepsy and Alzheimer's disease.

#### **PANNEXIN 1 (PANX1)**

Permeable to ions and small metabolites like ATP, Panx1 channels gained early notoriety as "death pores" in ischemic stroke and seizure (Thompson et al., 2006, 2008; Weilinger et al., 2012). Highly expressed in neonatal brain (Ray et al., 2005; Vogt et al., 2005), Panx1 also positively regulates proliferation and differentiation, and negatively regulates neurite outgrowth in developing neurons (Wicki-Stordeur et al., 2012; Wicki-Stordeur

*2 Department of Biology, University of Victoria, Victoria, BC, Canada*

and Swayne, 2013). Wicki-Stordeur and Swayne (2014) reviewed the growing Panx1 interactome to shed clues on the signaling pathways in which Panx1 might be involved, highlighting roles in cytoskeletal remodeling and inflammation.

## **MULTI-TASKING INTRACELLULAR MODULATORS**

A number of contributions underscore the capacity of "promiscuous" intracellular proteins to modulate a variety of ion channels and receptors through physical interaction. Reviewed by Donnelier and Braun (2014), cysteine string protein (CSP) is a resident pre-synaptic vesicle molecular chaperone targeting ion channels and vesicle-trafficking proteins. Not surprisingly, loss of, or mutation in CSP leads to synaptic dysfunction and neurodegeneration in a variety of systems (e.g., Zinsmaier et al., 1994; Fernandez-Chacon et al., 2004; Noskova et al., 2011). The sigma-1 receptor, reviewed by Pabba (2013), is an intracellular transmembrane protein that also acts in a chaperone-like way, modulating plasma membrane localized voltage- and ligand-gated channels with diverse neurophysiological and neuropathological implications. Harraz and Altier (2014) further link intracellular proteins to the regulation of plasma membrane channels, reviewing Stromal Interaction Molecule 1 (STIM1) in store-operated Ca2+ entry. They describe foundational work implicating STIM1 as the Ca2+ sensor in this process critical for maintaining neurotransmission. Further they outline key physical interactions between STIM1 with Ca2+-release activated channels and voltage-gated Ca2+ channels that coordinate the activation and inhibition of these types of channels, respectively. Finally, two papers by Wilson et al. (2014a,b) focus on another intracellular multifunctional/multi-interactome protein, collapsin response mediator protein 2 (Crmp2). Best known as a microtubule stabilizer, Crmp2 is regulated in a context specific way by multiple kinases, and in turn, positively regulates both ligand- and voltage-gated Ca2+ channels.

## **NEW FRONTIERS: TOWARD MORE COMPREHENSIVE MACROMOLECULAR NETWORKS**

Adding further complexity to ion channel networks is consideration of lipid membrane composition and lipid second messengers. In the sole lipidome-oriented contribution, Raboune et al. (2014) identify novel N-acyl amides regulating transient receptor potential vanilloid (TRPV) channels in the context of inflammatory pain. The future understanding of ion channel interactomes will undoubtedly include both proteome and lipidome components as technological advances in lipidomic research (Bou Khalil et al., 2010) become mainstream.

## **FINAL PERSPECTIVES: INTERACTOMES TO BEDSIDE**

While daunting, elucidating these macromolecular intricacies has a translational silver lining: while difficult to identify and unravel, the myriad interaction loci revealed by studying these interactions present unique opportunities for discrete, and potentially safer therapeutic intervention. For example, selective blockade at key interaction loci with cell-permeable peptides now provides an infinite number of ways in which interactomes can be discretely modulated to improve health outcomes.

## **REFERENCES**

activity-dependent increase in neurite outgrowth. *Front. Cell. Neurosci.* 8:196. doi: 10.3389/fncel.2014.00196

**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.

*Received: 26 September 2014; accepted: 10 November 2014; published online: 26 November 2014.*

*Citation: Swayne LA, Altier C and Zamponi GW (2014) The truth in complexes: perspectives on ion channel signaling nexuses in the nervous system. Front. Cell. Neurosci. 8:406. doi: 10.3389/fncel.2014.00406*

*This article was submitted to the journal Frontiers in Cellular Neuroscience.*

*Copyright © 2014 Swayne, Altier and Zamponi. 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) or licensor 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.*

## The NMDA receptor complex: a multifunctional machine at the glutamatergic synapse

## **Xuelai Fan, Wu Yang Jin and Yu Tian Wang\***

Brain Research Centre and Department of Medicine, Vancouver Coastal Health Research Institute, University of British Columbia, Vancouver, BC, Canada

#### **Edited by:**

Leigh Anne Swayne, University of Victoria, Canada

**Reviewed by:**

Carlo Sala, CNR Institute of Neuroscience, Italy Jean-Claude Beique, University of Ottawa, Canada

#### **\*Correspondence:**

Yu Tian Wang, Brain Research Centre and Department of Medicine, Vancouver Coastal Health Research Institute, University of British Columbia, 2211 Wesbrook Mall, Vancouver, BC V6T2B5, Canada e-mail<EMAIL_ADDRESS>
The N-methyl-D-aspartate receptors (NMDARs) are part of a large multiprotein complex at the glutamatergic synapse. The assembly of NMDARs with synaptic proteins offers a means to regulate NMDAR channel properties and receptor trafficking, and couples NMDAR activation to distinct intracellular signaling pathways, thus contributing to the versatility of NMDAR functions. Receptor-protein interactions at the synapse provide a dynamic and powerful mechanism for regulating synaptic efficacy, but can also contribute to NMDAR overactivation-induced excitotoxicity and cellular damage under pathological conditions. An emerging concept is that by understanding the mechanisms and functions of disease-specific protein-protein interactions in the NMDAR complex, we may be able to develop novel therapies based on protein-NMDAR interactions for the treatment of brain diseases in which NMDAR dysfunction is at the root of their pathogenesis.

**Keywords: NMDA receptors, protein-protein interaction, synaptic plasticity, dopamine receptors, excitotoxicity, ion channel complex, signaling pathways**

N-methyl-D-aspartate receptors (NMDARs) are a major subtype of glutamate-gated ion channels at the excitatory synapses in the central nervous system (CNS), which mediate the flow of sodium (Na+) and calcium (Ca2+) ions into the cell and potassium ions (K+) out of the cell. NMDARs are heterotetrameric plasma membrane channels composed of two obligatory GluN1 and two modulatory GluN2 (A-D) subunits (Cull-Candy et al., 2001; Collingridge et al., 2009), although sometimes the GluN2 subunits are replaced by GluN3(A-B) subunits (Ulbrich and Isacoff, 2008). NMDARs form a diheteromer when the two GluN2 subunits are identical, or a triheteromer when two different GluN2 subunits co-assemble with two identical GluN1 subunits (Ulbrich and Isacoff, 2008; Collingridge et al., 2010). At resting state, NMDARs are blocked by the presence of extracellular magnesium ions (Mg2+) in the channel pore (Vargas-Caballero and Robinson, 2004). As such, the activation and opening of NMDARs is both voltage-dependent and ligand-gated, and requires the binding of two ligands, glutamate and either D-serine or glycine (Nong et al., 2003; Papouin et al., 2012), at a depolarized membrane potential to relieve Mg2+ block. The function of NMDARs in the CNS has been extensively studied in both genetic and pharmacological manipulations. NMDARs play a critical role in a wide range of cellular processes and brain functions, including synaptic plasticity, addiction and stroke ( Schilström et al., 2006; Collingridge et al., 2010; Lai et al., 2011). The versatility of NMDAR functions may in part be attributed to its organization at the synapse. NMDARs are anchored to the plasma membrane as a multiprotein complex by binding to more than 70 adhesion proteins ( Naisbitt et al., 1999; Husi et al., 2000; Grant and O'Dell, 2001). Upon activation, Ca2+ influxes through the opened channel pore and triggers various intracellular signaling cascades by activating calcium-sensitive

NMDAR-interacting proteins in the multiprotein complex (Lai et al., 2011; Martin and Wellman, 2011; Lisman et al., 2012). Increasing evidence suggest that it is these interacting proteins that confer the versatile functions of NMDARs. In this review, we will introduce several key NMDAR-interacting proteins in the NMDAR multiprotein complex and discuss their critical roles in mediating physiological and pathological functions in the brain.

## **INTERACTIONS BETWEEN NMDARs AND CALCIUM SENSING PROTEINS IN SYNAPTIC PLASTICITY**

Two major forms of synaptic plasticity are long-term potentiation (LTP) and long-term depression (LTD), which are respectively characterized by long-lasting enhancement and reduction of synaptic transmission between two adjacent neurons after repetitive stimulation (Collingridge et al., 2010, 2004). Many proteins in the NMDAR complex contribute to these processes, with one of the most well-characterized proteins being Ca2+/calmodulindependent protein kinase II (CaMKII; Hayashi et al., 2000; Pi et al., 2010; Lisman et al., 2012), a serine/threonine protein kinase that is highly enriched in the post-synaptic density region. CaMKII is a large holoenzyme consisting of 12 identical subunits (Lisman et al., 2012). Transient Ca2+ influx through the NMDAR channel pore induces autophosphorylation of CaMKII (at T286 on CaMKIIα subunits or T287 on CaMKIIβ subunits), resulting in its persistent activation even after intracellular Ca2+ levels return to baseline (Rellos et al., 2010). As shown by a variety of real-time imaging studies and binding assays, activated CaMKII then rapidly and reversibly translocates to the spine and physically interacts with the 1260-1309aa domain in the carboxyl-terminal of the GluN2B NMDAR subunit (**Figure 1A**; Strack and Colbran, 1998; Bayer et al., 2001, 2006; Otmakhov et al., 2004; Zhang et al., 2008; Lisman et al., 2012). Although there is a basal level of association between CaMKII and NMDARs (Leonard et al., 1999), the translocation of these activated, phosphorylated CaMKII proteins greatly increases the total number of the kinase at the postsynaptic site. Introduction of a T286A mutation that prevents the autophosphorylation and activation of CaMKIIα in mice significantly impairs NMDAR-dependent LTP in the hippocampal CA1 area and memory performance in a Morris water maze task (Giese et al., 1998), suggesting that CaMKII plays a vital role in NMDAR-dependent synaptic plasticity. This is further supported by several other lines of evidence. For example, overexpressing a GluN2B carboxyl-terminal fragment (839-1482aa) that disrupts the physiological interaction between NMDAR/CaMKII leads to severe deficits in hippocampal LTP and spatial learning in transgenic mice (Zhou et al., 2007). LTP in organotypic hippocampal slices is impaired either by acute replacement of the synaptic GluN2B with GluN2A subunit that shows less binding affinity with CaMKII, or by expression of a mutant synaptic GluN2B subunit that markedly reduces the binding affinity with CaMKII (Barria and Malinow, 2005). Taken together, these results suggest that association of activated CaMKII and NMDARs may be a necessary step for NMDAR-dependent synaptic plasticity.

How the NMDAR-CaMKII interaction contributes to the production of LTP is still not fully understood. Several recent studies report that normal levels of LTP in the CA1 neurons in adult hippocampal slices can be induced after GluN2B-containing NMDARs are fully blocked, suggesting that functional activation of GluN2B-containing NMDARs may not be essential (Köhr et al., 2003; Liu et al., 2004; Woo et al., 2005; Foster et al., 2010). In this regard, it is interesting to note that a recent study has found that GluN2B in the NMDAR complex may function as a key scaffolding protein at excitatory synapses, and thus plays a critical role in LTP by recruiting molecules important for LTP through interacting with them via its cytoplasmic tail (Foster et al., 2010). Thus, it is likely that GluN2B may have a structural, rather than a functional, role for LTP production, presumably through the GluN2B-CaMKII interaction to recruit CaMKII to the activated synapses, and CaMKII (as a calcium-dependent kinase) in turn playing an essential role in LTP induction; however, the mechanistic details have not yet been elucidated. Bath application of AC3-1, a selective peptide inhibitor for CaMKII, to acute hippocampal slices prevents LTP induction, but has little effect on NMDAR channel function (Chen et al., 2001). This suggests that although activated CaMKII is recruited to the synaptic site by interacting with the GluN2B carboxyl-terminal, it does not regulate NMDAR channel function during synaptic plasticity. Indeed, several other studies have reported that activated CaMKII phosphorylates the S831 residue of the GluA1 subunit of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate glutamate receptor (AMPAR), a major glutamate receptor that mediates fast synaptic transmission, and increases the single-channel conductance of GluA1 containing AMPARs (**Figure 1A**; Barria et al., 1997; Derkach et al., 1999; Kristensen et al., 2011). This effect can be mimicked by mutating the S831 residue of GluA1 to the phosphomimic glutamate residue (Kristensen et al., 2011). Meanwhile, activated CaMKII can also phosphorylate stargazin (Tomita et al., 2005), an important postsynaptic scaffolding protein that facilitates the trafficking of AMPARs from the extrasynaptic space to the synaptic region (**Figure 1A**; Schnell et al., 2002; Tsui and Malenka, 2006; Opazo et al., 2010; but see Kessels et al., 2009). Taken together, these processes markedly enhance synaptic transmission so as to promote the expression of LTP, in particular the initial phase of LTP. Given that LTP can exist for hours or even weeks, yet CaMKII is generally inactivated in a relatively short timeframe (∼1 min) after transient synaptic stimulation (Lee et al., 2009), it is still unclear whether CaMKII plays any important roles in the maintenance of late-phase LTP. Furthermore, little is known about how activated CaMKII participates in LTD, the opposing form of synaptic plasticity to LTP, although several recent studies have suggested that autonomous CaMKII can lead to either LTP or LTD, depending on the phosphorylation state of the control point, T305/T306 (Pi et al., 2010; Coultrap et al., 2014). Answering these questions will further uncover details about the physiological functions of GluN2B-CaMKII interaction in the NMDAR multiprotein complex, and enhance our understanding of NMDARdependent synaptic plasticity.

Two other important calcium sensors in the NMDAR multiprotein complex are Ras-Guanine Nucleotide-Releasing Factor 1 (Ras-GRF1) and Ras-GRF2, a family of calcium-dependent guanine nucleotide exchange factors (GEF) that are predominantly expressed in adult CNS neurons (Feig, 2011). Structurally, Ras-GRF1 and Ras-GRF2 share many similarities, and contain several common functional domains, including the calmodulinbinding IQ domain, Ras GTPase–activating CDC25 domain and Rac GTPase-activating DH/PH domain (Feig, 2011). However, recent studies have shown that Ras-GRF1 and Ras-GRF2 interact with different NMDAR subunits and play strikingly different roles in NMDAR-dependent synaptic plasticity (**Figure 1B**; Feig, 2011). Knocking out Ras-GRF1 in mice shows minimal effects on both high frequency stimulation (HFS) and theta burst stimulation (TBS)-induced LTP in hippocampal slices, but leads to severe impairments in low frequency stimulation (LFS)-induced LTD (Li et al., 2006). In contrast, knocking out Ras-GRF2 significantly impairs both HFS and TBS-induced LTP in hippocampal slices, whereas LFS-induced LTD is not affected. More strikingly, as shown by immunoblotting studies in hippocampal brain slices, Ras-GRF2 mediates signaling from GluN2A-containing NMDARs to the Ras effector extracellular signal-related protein kinase 1/2 (Erk1/2) mitogen-activated protein (MAP) kinase, a promoter of LTP, whereas Ras-GRF1 mediates signaling from GluN2Bcontaining NMDARs to the Rac effector p38 MAP kinase, a promoter of LTD (**Figure 1B**; Li et al., 2006). Given that some evidence suggests that GluN2A-containing NMDARs promote LTP whereas GluN2B-containing NMDARs promote LTD (Liu et al., 2004; Massey et al., 2004), these distinct differences between Ras-GRF1 and Ras-GRF2 in synaptic plasticity may be partially explained by the selective interaction between Ras-GRF1 and the 886-1310aa domain in the carboxyl-terminal of GluN2B subunit (Krapivinsky et al., 2003). However, to date there is no clear evidence that supports the presence of a direct interaction between Ras-GRF2 and the GluN2A subunit, although it is suggested that Ras-GRF2 may localize in the vicinity of GluN2Acontaining NMDARs at the postsynaptic site (Jin and Feig, 2010).

Furthermore, given that both Ras-GRF1 and Ras-GRF2 contain the activation domains for both Ras and Rac GTPases, it is also important to determine how and why these two proteins are coupled with different downstream signaling cascades during LTP and LTD. One explanation is that the selective association between the synaptic Ras GTPase-activating protein SynGAP and GluN2B-containing NMDARs in the synapse may help inhibit Ras-GRF1 from activating the Ras/Erk signaling cascade (Kim et al., 2005). Alternatively, scaffolding proteins that selectively associate with either GluN2A or GluN2B may specifically target Ras-GRF2 and Ras-GRF1 to the Ras and Rac signaling cascades, respectively (Buchsbaum et al., 2002; Feig, 2011). However, the detailed mechanism has yet to be determined.

It is noteworthy that the contribution of Ras-GRFs to NMDAR-dependent synaptic plasticity is highly regulated by development in mice. Ras-GRF1 and Ras-GRF2 are only coupled to NMDARs in adult neurons beginning at 20 days of age in mice (Li et al., 2006), while the guanine nucleotide exchange factor Sos mediates NMDAR signaling in neurons derived from neonatal mice (Tian et al., 2004). As mentioned above (**Figure 1B**), previous studies report that beginning at 1 month of age, Ras-GRF1 preferentially mediates GluN2B-containing NMDAR-dependent LTD in the CA1 region of the hippocampus of mice via the Rac/p38 pathway (Li et al., 2006). Interestingly, beginning at 2 months of age in mice, the role of Ras-GRF1 dramatically shifts to support the induction of LTP, rather than LTD, in the CA1 region as a downstream effector of calcium-permeable, AMPA-type glutamate receptors (Jin et al., 2013). Surprisingly, this induction of LTP is also mediated by the Rac/p38 pathway, which was previously thought to be mainly associated with LTD (**Figure 1B**; Jin et al., 2013). It is still unclear how these signaling pathways switch during development, but further investigation is likely to reveal additional details on the regulatory mechanisms of Ras-GRF in synaptic plasticity at the NMDAR complex.

## **INTERACTIONS BETWEEN NMDARs AND G-PROTEIN COUPLED RECEPTORS (GPCRs) IN PSYCHIATRIC DISORDERS**

In addition to coupling to calcium sensing proteins, the NMDAR complex has extensive functional interactions with G-protein coupled receptors (GPCRs) through direct physical interaction. An example of this is the reciprocal modulation between NMDARs and dopamine receptors, a family of GPCRs that has been implicated in many psychiatric disorders, including schizophrenia (Seeman, 1987; Dolan et al., 1995).

Dopamine receptors comprise of five subtypes, D1R to D5R, which can be further classified pharmacologically into D1-like receptors consisting of D1R and D5R, and D2-like receptors consisting of D2R, D3R and D4R (Seeman, 1987; Tiberi et al., 1991; Dolan et al., 1995). Although earlier studies have shown presynaptic localization (Levey et al., 1993), it is now generally believed that D1-like receptors are strictly localized on cells that are postsynaptic to dopaminergic neurons (Hersch et al., 1995; Yung et al., 1995). In contrast, D2Rs and D3Rs are present both presynaptically and postsynaptically (Sokoloff et al., 2006). D1-like receptors increase intracellular cAMP concentrations through activation of the Gs/olf class of G proteins and subsequent

activation of adenylyl cyclase (AC), whereas D2-like receptors couple to the Gi/o class of G proteins to inhibit AC (Kebabian et al., 1972; Monsma et al., 1990). Numerous studies have demonstrated extensive crosstalk between DRs and NMDARs via direct physical association between the receptors (**Figure 2**, for a review, see Beaulieu and Gainetdinov, 2011).

D1Rs co-immunoprecipitates with both GluN1 and GluN2A subunits of the NMDAR in rat hippocampal tissue, suggesting a physical interaction between the two receptors (Lee et al., 2002). Further *in vitro* experiments confirm that the carboxyl tails of both the GluN1 subunit and GluN2A subunit (but not GluN2B) individually bind to the carboxyl tails of D1Rs (but not that of D5Rs). The 387-416aa domain of the D1R carboxyl-tail is sufficient for D1R-GluN1 binding, while the 417-446aa domain is required for the D1R-GluN2A interaction, suggesting that two distinct protein-protein interactions exist between the receptors. The binding between NMDARs and D1Rs occurs in the absence of D1R agonists, and D1R activation reduces the interaction between D1R-GluN1 but not that between D1R-GluN2A (Lee et al., 2002).

The functional consequences of these two interactions significantly differ. Following D1R stimulation with its agonist SKF81297, the D1R-GluN2A interaction functions to reduce the surface expression of NMDARs and hence the receptor-gated currents in both transfected cells and hippocampal neurons through a PKA/PKC independent pathway (Lee et al., 2002). It should be noted that SKF81297, in addition to activating D1Rs, has previously been shown to reduce NMDAR currents via a direct blockade of the NMDAR channel pore (**Figure 2A**, Cui et al., 2006). However, dissociation of D1R-GluN2A interaction using an interference peptide significantly reverses the attenuation of NMDAR currents, strongly arguing for a crucial role of the physical coupling between the two receptors, rather than a direct channel blockade, in mediating D1R activationinduced NMDAR inhibition (Lee et al., 2002). As overactivation of NMDAR is crucial for excitotoxic neuronal death (Lai et al., 2011), this reduction of surface expression of NMDARs would be expected to decrease Ca2+ influx and hence be neuroprotective. However, peptide-mediated dissociation of D1R-GluN2A does not affect neuronal survival following a NMDA insult. Instead, the uncoupling of D1R-GluN1 following D1R activation confers neuroprotection through the recruitment of calmodulin and phosphatidylinositol 3-kinase to GluN1, which activates cellsurvival signals (**Figure 2A**, Lee et al., 2002). These data suggest that D1R activation-induced inhibition of NMDAR-mediated currents and excitotoxicity are differentially mediated by distinct subunit-specific interactions in the NMDAR complex, further highlighting that specific protein-protein interactions dictate the functional outcomes of the NMDAR complex.

Further studies have demonstrated that D1R and NMDAR interactions may also reciprocally regulate receptor trafficking and surface expression in a NMDAR subunit-specific manner. D1Rs directly interact with the GluN1 subunit in cytoplasmic compartments, where they are retained, and the presence of the GluN2B subunit drives the translocation and insertion of the D1R-GluN1 receptor complex into the plasma membrane in medium spiny neurons and cotransfected cells (Fiorentini et al., 2003). This suggests that D1Rs and NMDARs are assembled as constitutive heteromeric complexes in cytoplasmic compartments prior delivery to functional sites, a process that does not depend on receptor activation (Fiorentini et al., 2003). Furthermore, constitutive association with GluN2B-containing NMDARs abolishes agonist-induced D1R internalization and stabilizes D1Rs at the post-synaptic density (Fiorentini et al., 2003). In contrast, although GluN2A-containing NMDARs can also recruit D1Rs to the cell surface, this effect depends on NMDAR stimulation. In cotransfected cells, activation of GluN2A-containing NMDARs increases their physical association with D1Rs, which drives the insertion of D1Rs into the plasma membrane (Pei et al., 2004). These differences in agonist dependency may perhaps be attributed to functional differences between GluN2A or GluN2Bcontaining NMDARs (for a review, see Traynelis et al., 2010). In addition to regulating D1R surface expression, NMDAR activation can also reduce D1R lateral diffusion in medium spiny neurons via increased GluN1-D1R interaction, which stabilizes D1Rs at the synapse (Scott et al., 2006). It should be noted, however, that the observed increase in D1R surface levels may be transient or restricted to certain neuronal populations *in vivo*, as a 90% reduction in GluN1 expression in mice did not impair striatal D1R pharmacology and function (Ramsey et al., 2008).

Conversely, the binding between D1Rs and NMDARs also modulates NMDAR surface dynamics at glutamatergic synapses, which offers a more direct means to regulate synaptic plasticity. D1R activation, which reduces D1R-GluN1 interaction at the perisynapse, allows NMDARs to laterally diffuse into the postsynaptic density where they favor LTP, an effect that was recapitulated by dissociating D1R-GluN1 binding with an interference peptide (Ladepeche et al., 2013). Dissociation of D1R-GluN1 upon D1R activation promotes CaMKII-GluN1 interaction and increases CaMKII activity, which in turn upregulates NMDARmediated LTP in primary hippocampal neurons and promotes spatial working memory in the delayed match-to-place version of the water maze in intact animals (Nai et al., 2010). While this effect may be cautiously interpreted as a result of physical dissociation between D1R-GluN1, it should be noted that D1R activation may also affect synaptic plasticity through protein phosphatase-dependent pathways (Frey et al., 1993; Stramiello and Wagner, 2008) as well as network mechanisms (Xu and Yao, 2010). However, the relative contribution of each mechanism remains to be seen.

The long-form D2R, which is preferentially involved in postsynaptic signaling compared to short-form D2R (Lindgren et al., 2003) interacts with the carboxyl-tail of the GluN2B subunit of NMDARs in the post-synaptic density of striatal neurons. In contrast to D1Rs, this interaction is not mediated by the D2R carboxyl-tail, but by a TKRSSRAFRA motif situated in the N-terminal of the third intracellular loop of D2Rs (Liu et al., 2006). The interaction between D2Rs and NMDARs is receptorand subunit- specific: D3Rs, which are similar to D2Rs and also belong to the D2-class of dopamine receptors, do not associate with the GluN2B subunit, and D2Rs do not interact with the GluN1 subunit (Liu et al., 2006).

Activation of D2Rs with its agonist quinpirole inhibits NMDAR currents in acutely dissociated medium-sized striatal neurons, which can be blocked by disrupting D2R-GluN2B physical interaction (Liu et al., 2006). Acute treatment with cocaine, a psychostimulant known to exert its effects through both dopaminergic and glutamatergic signaling, enhances D2R-GluN2B coupling in the striatum. In turn, this increased association disrupts GluN2B-CaMKII binding, resulting in decreased CaMKII activity, reducing phosphorylation of GluN2B at the Serine 1303 residue, and thereby resulting in reduced NMDAR currents (**Figure 2B**, Liu et al., 2006). Cocaine-enhanced D2R-GluN2B interaction seems to be specific to the striatum, as it was not observed in the hippocampus or frontal cortex. The D2R-GluN2B interaction may play a role in eliciting the locomotor effects of cocaine. In rats, systemic disruption of D2R-GluN2B interaction prior cocaine treatment markedly reverses the uncoupling between GluN2B-CaMKII induced by cocaine and rescues GluN2B S1303 phosphorylation in the striatum without affecting basal GluN2B-CaMKII interactions. Furthermore, blocking D2R-GluN2B interactions significantly, though not completely, reduces cocaine-stimulated locomotion in rats (Liu et al., 2006). This suggests the existence of other factors that contribute to the fullscale motor response to cocaine.

Going forward, it would be particularly interesting to see whether these findings extend to prefrontal dopaminergic neurotransmission. The prefrontal cortex, together with other corticolimbic areas including the cingulate gyrus and hippocampus, are implicated as part of the dysfunctional network that underlies schizophrenia (Fletcher, 1998). Furthermore, NMDAR hypofunction during development is gradually emerging as a convergence point for disease progression in schizophrenia (Snyder and Gao, 2013). Together, these findings, with the observation that the D1R-GluN1 physical interaction reciprocally regulates D1R and NMDAR surface expression and function, prompts the question of whether it is the initial failure in glutamatergic signaling that leads to reduced dopaminergic neurotransmission in schizophrenia through perturbed protein (receptor)-NMDAR interactions in the NMDAR complex.

In summary, NDMARs and dopamine receptors directly interact to reciprocally regulate receptor surface expression, channel properties and downstream intracellular signaling cascades. Together with functional interactions through shared downstream signaling molecules, the physical coupling between NMDARs and dopamine receptors adds another layer of regulation to both neurotransmitter systems to fine-tune neuronal function and behavior.

## **NMDAR COMPLEX AS A CRUX IN ISCHEMIC NEURONAL DAMAGES**

Intensive investigations into the mechanisms and functions of protein-protein interactions in the NMDAR complex have not only further advanced our understanding of the roles of NMDARs in brain function and dysfunction, but also led to the development of novel protein-NMDAR interaction-based therapeutics for treating brain disorders in which NMDAR dysfunction is at the root of their pathogenesis. Well-characterized examples of such therapeutics are the newly developed and promising interventions that protect neurons against excitotoxic/ischemic damages following stroke by disrupting direct or indirect interactions between NMDARs and neuronal death signaling molecules, such as neuronal Nitric Oxide Synthase (nNOS; Aarts et al., 2002; Zhou et al., 2010), death associated protein kinase 1 (DAPK1; Tu et al., 2010; Fan et al., 2014) and PTEN (Zhang et al., 2013).

Overactivation of the NMDAR triggers rapid Ca2+ influx that could lead to excitotoxic neuronal death. This excitotoxicity is considered a common pathological step leading to neuronal

loss in many brain disorders, from acute brain injuries such as stroke to chronic neurodegenerative diseases such as Huntington's disease (Lai et al., 2014). Yet direct blockade of NMDARs has failed as a neuroprotective strategy in stroke, at least in part due to intolerable psychosomatic side effects as a result of blocking normal NMDAR function and/or relatively narrow windows for effective intervention (Ikonomidou and Turski, 2002; Lai et al., 2014). In an effort to overcome the shortcomings of NMDAR antagonists, scientists have focused on druggable protein-protein interactions in the NMDAR complex that specifically lead to neuronal death. These have led to not only the identification of several well-characterized cell death-promoting molecules that can form neuronal death signaling complexes downstream of NMDARs via either direct or indirect interactions with NMDAR subunits, but also the development of numerous promising interventions that protect neurons from brain insults by specifically disrupting these interactions (Lai et al., 2014).

One of the most exciting bench-to-bedside examples is the recent development of effective neuroprotectants based on disrupting the GluN2B-Post synaptic density-95 (PSD-95)-nNOS cell death complex (Christopherson, 1999; Sattler et al., 1999), which was first characterized over a decade ago. PSD-95 is a membrane-associated guanylate kinase (MAGUK) concentrated at glutamatergic synapses and is involved in synapse stabilization and plasticity (El-Husseini et al., 2000). nNOS catalyzes the production of nitric oxide (NO), a diffusible signaling molecule implicated in synaptic plasticity (Bredt et al., 1990) and glutamate neurotoxicity (Dawson et al., 1991). By binding to both nNOS and GluN2B through its different PDZ domains, PSD-95 functions as a scaffolding protein to bring nNOS to the NMDAR complex (Kornau et al., 1995; Brenman et al., 1996). Following excitotoxic stimuli and/or ischemic insults, PSD-95 tethers nNOS to GluN2B, thus positioning nNOS to be more effectively activated by Ca2+ influx through the NMDAR channel pore and generation of the cytotoxic compound NO (**Figure 3**, left panel) (Dawson et al., 1991; Sattler et al., 1999).

In a proof-of-concept study, Aarts et al. (2002) disrupted this signaling complex with a 20-mer interference peptide Tat-NR2B9c, which comprises of the last nine amino acids of the carboxyl tail of GluN2B required for its interaction with PSD-95 and the 11-mer Tat protein transduction domain that renders the peptide plasma membrane permeable. The rationale behind the initial peptide design is to use it to disrupt GluN2B-PSD-95 interaction and hence to prevent PSD-95 from recruiting nNOS to the NMDAR complex, thereby reducing NO production and NO-mediated neuronal death (**Figure 3**, middle panel), although a later study suggests that the peptide may also somehow reduce the interaction between PSD-95 and nNOS (Cui et al., 2007). When bath applied to primary neuronal cultures and acute brain slices, Tat-NR2B9c does not affect NMDARmediated currents or Ca2+ fluxes, but can selectively disrupt the interaction between PSD-95 and NMDARs through competition with the binding of native GluN2B subunit (but not GluN2A) to PSD95, and thereby reduce the generation of NO and excitotoxicity (Aarts et al., 2002). When given systematically, a single dose of Tat-NR2B9c administered either before or after ischemic insults reduces ischemic brain damage with concurrent improvements in neurological scores in rats subjected to transient middle cerebral artery occlusion (MCAo), a wellcharacterized focal ischemia stroke model (Aarts et al., 2002). Similar neuroprotective effects are seen by disrupting the interaction between PSD-95 and nNOS with an interference peptide derived from the PSD-95 binding domain of nNOS (nNOS-N1−133; **Figure 3**, right panel; Zhou et al., 2010). Following viral infection of rats with a vector expressing this peptide, nNOS-N1−133 effectively blocks the interaction between the two proteins and significantly reduces stroke-induced ischemic damage (Zhou et al., 2010).

These initial attempts at targeting GluN2B-PSD-95-nNOS signaling pathway for the treatment of stroke was quickly followed by a well-designed study in a focal ischemia model in gyrencephalic nonhuman primates (Cook et al., 2012a,b) and a successful phase 2, double-blind, placebo-controlled multicenter clinical trial of neuroprotection in procedurally-induced stroke (Hill et al., 2012). Treatment with Tat-NR2B9c (also named as NA-1 in these studies) results in reduced ischemic brain damage compared to the placebo treatment, as evidenced by diffusion weighted magnetic resonance imaging. These exciting bench-tobedside results provide great promises for further understanding disease-specific protein-protein interactions in the NMDAR complex and thereby developing novel and effective therapeutics for brain diseases involving NMDAR-mediated neuronal degeneration.

### **CONCLUSIONS**

Increasing evidence suggest that the NMDAR is not a solo player in the regulation of many brain functions and dysfunctions. By associating with various membrane receptors and extracellular or intracellular proteins in the complex, the NMDAR can contribute to various physiological processes such as learning and memory, and brain disorders such as stroke, schizophrenia and addiction. Many NMDAR-interacting proteins have recently been identified in the NMDAR complex; however, in the past only a few protein-protein interactions have been characterized in detail. Since NMDARs are widely expressed in all brain areas, it would be paramount to know whether and how the NMDAR plays vital roles in both brain function and dysfunction through these diverse protein-protein interactions in the receptor complex. Further investigation on this topic will not only deepen our understanding of the functions of the NMDAR multiprotein complex, but also greatly facilitate the development of innovative therapeutics in treating various NMDAR dysfunction-related brain disorders.

#### **ACKNOWLEDGMENTS**

We thank Dr. Loren Oschipok for his excellent editorial assistance. This work was in part supported by the Canadian Institutes of Health Research (CIHR). Yu Tian Wang is the holder of Heart and Stroke Foundation of British Columbia and Yukon Chair in Stroke Research.

#### **REFERENCES**

**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.

*Received: 13 April 2014; accepted: 26 May 2014; published online: 10 June 2014*. *Citation: Fan X, Jin WY and Wang YT (2014) The NMDA receptor complex: a multifunctional machine at the glutamatergic synapse. Front. Cell. Neurosci. 8:160. doi: 10.3389/fncel.2014.00160*

*This article was submitted to the journal Frontiers in Cellular Neuroscience*.

*Copyright © 2014 Fan, Jin and Wang. 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) or licensor 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.*

## Ligand-gated ion channel interacting proteins and their role in neuroprotection

## *Shupeng Li , Albert H. C. Wong and Fang Liu\**

*Campbell Family Mental Health Research Institute, Centre for Addiction and Mental Health, University of Toronto, Toronto, ON, Canada*

#### *Edited by:*

*Gerald Zamponi, University of Calgary, Canada*

#### *Reviewed by:*

*Laurent Fagni, University of Montpellier, France Stephen Ferguson, Western, Canada*

#### *\*Correspondence:*

*Fang Liu, Centre for Addiction and Mental Health, 250 College Street, Toronto, ON M5T 1R8, Canada e-mail<EMAIL_ADDRESS>
Ion channel receptors are a vital component of nervous system signaling, allowing rapid and direct conversion of a chemical neurotransmitter message to an electrical current. In recent decades, it has become apparent that ionotropic receptors are regulated by protein-protein interactions with other ion channels, G-protein coupled receptors and intracellular proteins. These other proteins can also be modulated by these interactions with ion channel receptors. This bidirectional functional cross-talk is important for critical cellular functions such as excitotoxicity in pathological and disease states like stroke, and for the basic dynamics of activity-dependent synaptic plasticity. Protein interactions with ion channel receptors can therefore increase the computational capacity of neuronal signaling cascades and also represent a novel target for therapeutic intervention in neuropsychiatric disease. This review will highlight some examples of ion channel receptor interactions and their potential clinical utility for neuroprotection.

**Keywords: ligand-gated ion channels, protein-protein interaction, receptor complex, glutamate receptors, dopamine receptors**

Efficient neurotransmission requires the precise interplay of various neurotransmitter receptors at pre- and post-synaptic compartments. Ligand-gated ion channels play a central role in intercellular communication in the nervous system. Ion channels are the cellular machinery for ion flux across the membrane and therefore the basis of electrical excitation of neurons. Ligandgated ion channels are oligomeric protein assemblies that convert a chemical signal into an ion flux through the post-synaptic membrane, and are involved in basic brain functions such as attention, learning, and memory (Ashcroft, 2006). This paper will review some interactions between ionotropic receptors and other proteins that affect signaling, metabolism, and trafficking. We will detail some of the interactions between G-protein coupled receptors and ion channels, ion channels with intracellular proteins, and ion channels with other ion channels. Specifically, we will discuss how these receptor-protein interactions may help to understand pathology of the nervous system, and offer the hope of new treatment targets for neuropsychiatric disease.

Most eukaryotic membrane channels are composed of several subunits. Pentameric ligand-gated ion channels have five subunits arranged in a pseudosymmetric rosette around the centrally located ion channel pore. Each subunit consists of an extracellular domain that contains the ligand-binding site and a transmembrane ion-pore domain (Kozuska and Paulsen, 2012). The agonist binding site is located at the boundary between subunits in the extracellular domain whereas the ion pore is formed by the transmembrane helices M2, which differentiate axial cationic and anionic channels (Dacosta and Baenziger, 2013).

The modular construction of ionotropic receptors increases the diversity of ligand binding and ion selectivity through the mixing and matching of various subunits (Rojas and Dingledine, 2013). Various other proteins are involved in controlling the type and position of particular subunits in a given channel, as well as the function of the function of the assembled channel. Thus, the function of many receptor proteins can only be fully understood in the context of their interactions with other proteins, the "interactome" (Li et al., 2013a). Through this extensive protein network, the binding of agonist to an ionotropic receptor can exert effects beyond simple current flow, including gene transcription, cytoskeletal rearrangement, and protein synthesis, degradation and transport.

The ligand-gated ion channel superfamily includes nicotinic acetylcholine receptors (nAChRs), adenosine triphosphate (ATP) receptors, γ-aminobutyric acid (GABA), glutamate, glycine, and 5-hydroxytryptamine (5-HT) receptors (Dent, 2010). The "Cys-loop" sub-family includes the ionotropic receptors for nicotine, GABA, glycine, and 5-HT. "Pore-loop" channels include inwardly-rectifying potassium channels and the glutamate receptors of the AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) subtype and NMDA (N-methyl-Daspartate) receptors (Connolly and Wafford, 2004). The structure and basic features of several important ion channel types is briefly reviewed below to provide some background, before discussing ion channel interactions with other proteins.

AMPA receptors are the major mediator of fast excitatory synaptic transmission in the mammalian central nervous system. Most of these ionotropic glutamate receptors are permeable to calcium and other cations, thus facilitating depolarization of the cell membrane. AMPA receptors contain four subunits, consisting of two dimers formed by combinations of four pore-forming subunits GluA 1–4. GluA 1/2, and GluA 2/3 are the predominant AMPA receptor types (Anggono and Huganir, 2012). Each subunit is an integral membrane protein with an extracellular amino terminal domain, three membrane-spanning domains (M1, M3, and M4), a hydrophobic hairpin domain (M2) that forms the channel pore, and three intracellular domains: Loop1, Loop2, and the carboxyl tail. Although the carboxyl tail is crucial for receptor trafficking and function, other intracellular domains also play an important role in the regulation of AMPA receptor trafficking (Rojas and Dingledine, 2013). The pore lining domain also influences the AMPA receptor trafficking, early in the secretory pathway.

NMDA receptors are also ionotropic glutamate receptors permeable to calcium and sodium, and are critical for synaptic plasticity, learning, and memory. These receptors are involved nicotine addiction and neurological disorders such as ischemic stroke (Dingledine et al., 1999; Albensi, 2007). NMDA channel opening is gated by several elements, including the binding of glutamate to the NR2 subunit, a co-ligand glycine which binds to a separate site on the NR1 subunit, and the removal of a magnesium ion block by depolarization. Thus NMDA channels require both agonist stimulation and depolarization of the post-synaptic membrane for current flow, effectively detecting coincident pre- and post-synaptic activation. Like AMPA receptors, NMDA receptors consist of four subunits, two of which are obligatory GluN1 and two which are modulatory GluN2. These subunits are present in various isoforms that in the NR1 subunit are generated by alternative splicing of the GRIN1 gene. The GluN2 subunits are each encoded by different genes and the subunits are named: NR2A, B, C, and D (Durand et al., 1992; Sugihara et al., 1992; Hollmann et al., 1993; Michaelis, 1998). The exact subunit composition of native NMDARs remains unknown, but it has been suggested that native NMDARs in rat hippocampus largely contain NR1/2A subunits. The intracellular C-terminal (Allison et al., 1998) contains potential sites of protein phosphorylation by different kinases, and motifs for interacting with various protein partners.

For pentameric ligand-gated ion channels such as the GABAA receptor, each subunit contains an extensive N-terminal extracellular domain and a short extracellular C-terminal sequence containing the ligand binding sites. Structurally, each receptor is composed of four closely-spaced transmembrane domains including the ion channel wall primarily in M2, and a variable length cytoplasmic loop between M3 and M4. With both the N-terminus and C-terminus of receptor subunits extending outside the cell membrane, the intracellular M3-M4 loop becomes the most important domain interacting with the intracellular environment. The M3-M4 intracellular loop (IL2) contains protein-protein interaction domains involved in regulating synaptic localization and intracellular trafficking (Maksay, 2009). Despite being highly variable in length and amino acid sequence, the IL2 loop usually contains numerous regulatory motifs and binding sites that differ in each subunit isoform. Moreover, this IL2 loop usually contains sites for phosphorylation, palmitoylation, ubiquitination and other modifications that modulate protein interactions and eventually determine the clustering, stability, and function of ligand-gated ion channels.

Within the central nervous system (CNS), nicotine acts on nicotinic receptors (nAChRs) to initiate various physiological and pathological processes at the cellular and circuit level. Similar to the NMDAR, nAChR is also a ligand-gated ion channel receptor with high Ca2+ permeability. The pentameric nAChR has a number of different subtypes, each with individual pharmacological and physiological profiles and a distinct anatomical distribution in the brain (Gotti et al., 2009). There are 11 neuronal nAChR subunits identified in mammals, including eight α(α2 –α9) and three β (β2– β4). Unlike NMDAR, nAChR can exist as both hetero-metric and homo-metric-assemblies of these subunits (Albuquerque et al., 2009). In rodents, the β2 subunit is found throughout the CNS, mostly as part of the α4β2 heteromeric receptor, which exhibits high affinity for nicotine. The second common AChR isoform is the α7 homopentamer, expressed mainly in the cortex, hippocampus, and limbic areas.

Numerous proteins are known to bind to the different subunits of pentameric ligand-gated ion channel receptors at specific subcellular localizations. These interacting proteins can both modulate ion channel function and influence the localization of the channel in the cell and in the membrane. There are several major categories of proteins that ligand-gated ion channels interact with: G-protein coupled receptors, intracellular proteins, and other ligand-gated ion channels (Kittler and Moss, 2003; Li et al., 2012; Rojas and Dingledine, 2013). We will discuss each of these types of interaction with examples drawn from our previous work.

## **LIGAND-GATED ION CHANNEL INTERACTIONS WITH G-PROTEIN COUPLED RECEPTORS**

There are several ion channel interactions with G-protein coupled receptors reported by our group, all involving dopamine receptors: D5-GABAA (Liu et al., 2000), D1-NMDA (Lee et al., 2002; Pei et al., 2004), and D2-AMPA (Zou et al., 2005).

First discovered in 2000, the interaction between γaminobutyric acid A receptors and the dopamine D5 receptor was one of the earliest examples of interactions between metabotropic and ionotropic receptors (Liu et al., 2000). The GABAA receptor is the major inhibitory neurotransmitter receptor that conducts chloride ions, resulting in hyperpolarization of the cell membrane and thus inhibiting neuron depolarization. The second intracellular loop of the GABAA γ2 (short) receptor subunit binds directly to the carboxy-terminus of the D5 receptors. This interaction reduces D5 receptor-mediated cAMP accumulation without altering affinity of the D5 for endogenous and experimental ligands. Conversely, activation of D5 receptors reduces GABAA currents and this is not dependent on PKC or PKA. Because these two receptors co-localize in hippocampal and cortical neurons, the bi-directional modulation is potentially important for a number of behaviors and brain disorders. A notable feature of this reciprocal modulation is that it is independent of the classical signaling pathways for each individual receptor.

The interaction between the D1 and NMDA receptor involves direct binding between the carboxy termini of the D1 receptor and the NR1-1a and NR2A subunits of the NMDA receptor (Lee et al., 2002). Because there are two interacting components in the NMDA receptor, the functional consequences are more complicated than with the D5-GABAA interaction. Although the D1-NMDA interaction occurs in the absence of D1 receptor agonist stimulation, the D1 agonist SKF 81297 decreases the amount of D1-NMDA complex. However, the agonist affects only D1-NR1-1a binding, not the D1-NR2A interaction.

Similar to the D5-GABAA interaction, D1 agonist stimulation reduces NMDA currents as well as reducing the slope of the current-voltage curve without altering the reversal potential. This decrease in NMDA conductance is accompanied by a reduction in NMDA cell surface expression. It is independent of PKA/PKC pathways and is mediated by the D1 interaction with the NR2A subunit. The D1-NR1-1a interaction mediates neuroprotective effects of D1 receptor activation, and these effects are not dependent on G-protein signaling but rather are dependent on a PI-3 kinase mechanism. The proposed model is that agonist activation of D1 receptors promotes dissociation of the D1-NR1-1a interaction, allowing the NR1-1a carboxy tail to bind CaM and PI-3 kinase, thereby initiating protective cellular cascades. Conversely, the D1 receptor is also regulated by NMDA receptor activation, which increases cell surface D1 receptor expression, and enhances cAMP accumulation in a SNARE-dependent fashion (Pei et al., 2004).

Dopamine D1 receptors also have a direct physical interaction with N-type calcium channels (Kisilevsky et al., 2008). In prefrontal cortex neurons dopamine D1 receptor activation reduced calcium influx through voltage-gated calcium channels, likely through both voltage-dependent G-protein-mediated pathways and through PKA-mediated pathways. The D1-Cav2.2 channel interaction also regulates cell surface expression and distribution of the channel, allowing effective dopaminergic regulation of backpropogating action potentials that are mediated by N-type calcium channels.

## **LIGAND-GATED ION CHANNEL INTERACTIONS WITH INTRACELLULAR PROTEINS**

The second category of ion channel interactions to be considered is with intracellular proteins. For the GABAA receptor, multiple interacting proteins have been characterized, that play a role in cytoskeletal coupling by gephyrin, radixin, and F-actin and in signaling modulation by RAFT1 and collybistin (Chen and Olsen, 2007). The functional characteristics of this modulation are related to their subcellular localization with receptors. For example, GABARAP, GODZ, and Plic1 interact with the intracellular parts of the GABAA receptor, while GRIP1 and gephyrin are localized to sub-membrane and intracellular compartments.

Our group has recently reported an interaction between the N-terminus of the GluR2 sub-unit of the AMPA receptor and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Wang et al., 2012; Zhai et al., 2013). GAPDH is a metabolic enzyme that participates in glycolysis and also plays a role in apoptosis (Tarze et al., 2007). AMPA receptor activation by agonist increases formation of the GluR2-GAPDH complex and promotes cellular internalization. Blocking this interaction with a peptide protects cells against glutamatergic excitotoxicity and ischemia-induced neuronal damage. This interaction is therefore of potential clinical interest for the treatment of ischemic stroke or other conditions with neuronal damage.

Another ion channel-protein interaction that has been of interest because of neuroprotective effects is between the PDZ domain of post-synaptic density 95 (PSD95) protein and carboxyl terminus of the NMDA NR2 subunit (Kornau et al., 1995; Niethammer et al., 1996; Cui et al., 2007). This interaction couples NMDA receptor activation to nitric oxide neurotoxicity (Sattler et al., 1999), and disrupting this interaction can protect neurons from excitotoxicity and ischemic brain damage both *in vitro* and *in vivo* (Aarts et al., 2002). PSD-95 also interacts with and suppresses the tyrosine kinase Src and attenuates Src-mediated NMDA receptor upregulation (Kalia et al., 2006). Consistent with these findings, inhibitors of PSD-95 also show neuroprotective effects in animal models of stroke (Sun et al., 2008).

While several examples of direct interactions between ion channels and G-protein coupled receptors have been discussed above, these two types of receptors can also exert functional crosstalk through indirect interactions. For example, the presynaptic voltage-gated calcium channels that influence neurotransmitter release are regulated by G-protein activation and protein kinase C-dependent phosphorylation through binding to Gβγ (Zamponi et al., 1997). G-protein modulation of N-type calcium channels also involves syntaxin 1A, a member of the SNARE protein complex responsible for synaptic vesicle fusion during neurotransmitter release (Jarvis et al., 2000). An additional modulator is cysteine string protein or CSP, which also bind to N-type calcium channels in conjunction with G-proteins to exert a tonic inhibition of the channel (Magga et al., 2000). In the case of G-protein activation in inwardly-rectifying potassium channels (GIRK), the Gβγ directly gates ion channel opening by binding to the intracellular pore of the channel (Nishida and MacKinnon, 2002).

## **LIGAND-GATED ION CHANNEL INTERACTIONS WITH OTHER ION CHANNELS**

Ion channel receptors can also interact with other ion channels, such as the interaction between the α7 nicotinic acetylcholine receptors and NMDA receptors (α7nAChR-NMDA) (Li et al., 2012, 2013b). The carboxy tail of the NMDA receptor NR2 subunit binds directly with the second intracellular loop of the α7nACh receptor, and the interaction promotes ERK1/2 phosphorylation. This interaction is of clinical interest since nicotine increases formation of the complex, and disrupting the α7nAChR-NMDA interaction blocks cue-induced reinstatement of nicotine self-administration in the rat. This behavioral test is a model of relapse in nicotine addiction, suggesting that the α7nAChR-NMDA interaction could be a useful target for novel smoking cessation therapies.

## **TARGETING LIGAND-GATED ION CHANNEL INTERACTIONS FOR NEUROPROTECTION**

Because of the involvement of ion channel receptors in neuronal death from excitatory glutamate stimulation, there has been considerable interest in these receptors as therapeutic targets for the treatment of brain disorders involving neuronal death, such as ischemic stroke. Ischemic stroke is a major medical problem that affects millions of people world-wide. Current acute post-stroke treatment is focused on lysing the clot obstructing arterial blood flow by using a tissue plasminogen-activator. Due to a very short time window for effectiveness and the potential for intracranial bleeding, few patients can benefit from this treatment (Grossman and Broderick, 2013). Therefore, there is a major need for new and safer drugs that can reduce the extent of brain injury from ischemic stroke.

An alternative strategy for post-stroke treatment is to target neurotoxicity instead of focusing on the blood vessel blockade, or in addition to clot lysis. However, preventing excitotoxicity is difficult because glutamate receptors have a critical role in many brain functions. AMPA/kainate receptor antagonists such as NBQX or MPQX can reduce neurological deficits in animal models of autoimmune damage (Smith et al., 2000), but these drugs are too toxic for clinical use. Other strategies, such as blocking the glycine site of the NMDA receptor for treating ischemic stroke have been ineffective in improving outcomes (Lees et al., 2000; Sacco et al., 2001).

The interactions between ionic glutamate receptors and other proteins such as GluR2-GAPDH and NR2-PSD-95 can improve cell survival after ischemic insults, and thus represent another approach to neuroprotective treatments after stroke (Sattler et al., 1999; Zhai et al., 2013). This strategy is attractive because the basic signal transducing functions of the channels are not blocked as they would be by a conventional antagonist. Thus, a more subtle modulation of ion channel function can be achieved, with the hope of reducing excitotoxicity while sparing normal neurotransmission. Those *in vitro* and animal model experiments used small interfering peptides to disrupt the channel-protein interactions, but these peptides may not be ideal for human clinical use. Therefore, an important priority for future research is the development of suitable drugs targeting the ion channel-protein interactions involved in excitotoxicity.

In summary, ion channel function is modulated by interactions with other proteins. Various proteins influence the number and position of particular subunits in the assembled channel, the dynamics of receptor trafficking and targeting to designated subcellular areas, as well as the assembly, stability, and turnover of the receptor on the membrane and in intracellular signaling pathways. These interactions permit the function of ion channels to be fine-tuned according to both external stimuli and intracellular states. This network of molecular interactions allows for complex signal processing by neurons and also reveals new targets for pharmacological manipulation of neuron function that may be clinically useful. Knowledge of ion channel interactions has progressed rapidly and more such interactions are likely to emerge.

## **REFERENCES**

**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.

*Received: 14 March 2014; accepted: 21 April 2014; published online: 09 May 2014. Citation: Li S, Wong AHC and Liu F (2014) Ligand-gated ion channel interacting proteins and their role in neuroprotection. Front. Cell. Neurosci. 8:125. doi: 10.3389/ fncel.2014.00125*

*This article was submitted to the journal Frontiers in Cellular Neuroscience.*

*Copyright © 2014 Li, Wong and Liu. 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) or licensor 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.*

## Kir3 channel ontogeny – the role of Gβγ subunits in channel assembly and trafficking

## *Peter Zylbergold1 †, Rory Sleno1 †, Shahriar M. Khan1, Ashley M. Jacobi 2 , Mark A. Belhke2 and Terence E. Hébert1\**

*1 Department of Pharmacology and Therapeutics, McGill University, Montréal, QC, Canada*

*2 Integrated DNA Technologies, Inc., Coralville, IA, USA*

#### *Edited by:*

*Gerald Zamponi, University of Calgary, Canada*

#### *Reviewed by:*

*Leigh Anne Swayne, University of Victoria, Canada Christophe Altier, University of Calgary, Canada*

#### *\*Correspondence:*

*Terence E. Hébert, Department of Pharmacology and Therapeutics, McGill University, 3655 Promenade Sir-William-Osler, Room 1303, Montréal, QC H3G 1Y6, Canada e-mail<EMAIL_ADDRESS>
†*Peter Zylbergold and Rory Sleno have contributed equally to this work.*

**INTRODUCTION**

Kir3 channels were first discovered in the context of the KAChchannel (Kir3.1/Kir3.4), activated by the muscarinic acetylcholine receptor expressed in the heart. Logothetis et al. (1987) first noted that hyperpolarization of chick embryonic atrial cells via the iKACh depended on Gβγ but not Gα subunits. It was later proposed that Gβγ acted on Kir3 through direct interactions with the channel (Huang et al., 1995; Inanobe et al., 1995; Krapivinsky et al., 1995b) and that different interacting domains underlie basal and agonist-induced activation. Specifically, it was shown that Leu339 on Kir3.4 and its counterpart Leu333 on Kir3.1 were critical for receptor-stimulated currents and that mutation of these residues to Glu completely abrogated channel activation (He et al., 1999). Surprisingly, these mutations did nothing to basal current, suggesting that distinct regions on the channel allowed Gβγ to facilitate basal activity. It was shown that both the N- (aa 34–86) and C-termini of Kir3.1 could bind Gβγ and that there was in fact two distinct binding domains on the C-terminus (aa 318–374 and aa 390–462), conferring greater Gβγ binding when Kir3.1 was part of the tetrameric channel (Huang et al., 1997). They also showed that the N- and C-termini of Kir3 channels interacted with each other and that when the C-terminus of Kir3.1 interacted with either the N-terminus of Kir3.1 or Kir3.4, substantial increases in Gβγ binding were observed (Huang et al., 1997). It was later found that mutation of single residues His57 and Leu262 on Kir3.1 (His64 and Leu268 on Kir3.4, respectively) were sufficient to reduce Gβγ activation of the channel complex (He et al., 2002). Glutathione-S-transferase-pull down experiments and competition assays demonstrated critical Gβγ binding domains on the Kir3.2 subunit. Using Kir3.1 and Kir3.2

The role of Gβγ subunits in Kir3 channel gating is well characterized. Here, we have studied the role of Gβγ dimers during their initial contact with Kir3 channels, prior to their insertion into the plasma membrane. We show that distinct Gβγ subunits play an important role in orchestrating and fine-tuning parts of the Kir3 channel life cycle. Gβ1γ2, apart from its role in channel opening that it shares with other Gβγ subunit combinations, may play a unique role in protecting maturing channels from degradation as they transit to the cell surface. Taken together, our data suggest that Gβ1γ2 prolongs the lifetime of the Kir3.1/Kir3.2 heterotetramer, although further studies would be required to shed more light on these early Gβγ effects on Kir3 maturation and trafficking.

**Keywords: Kir3 channels, G proteins, G protein-coupled inwardly-rectifying potassium channels, Gβγ subunits, channel assembly**

> as templates, Ivanina et al. (2003) confirmed that similar regions of the N-terminus of the two subunits bound Gβγ, whereas significant differences were found at the level of the C-terminus. Consistent with Huang et al. (1997) Kir3.1 revealed two distinct Gβγ binding domains on its C-terminus, one proximal and one distal, while the Kir3.2 subunit possessed only one C-terminal binding site at its most distal end (Ivanina et al., 2003). Interestingly, mutation of these C-terminal sites on the Kir3.1 subunit, both proximal and distal, did little to the binding of Gβγ to the channel, but dramatically altered the current characteristics, suggesting these interacting domains were more involved in channel gating dynamics rather than Gβγ binding (Ivanina et al., 2003).

> It was initially thought that Kir3 channels, like other effectors for G protein-coupled receptors (GPCRs), were trafficked independently of their cognate receptors, G proteins and auxiliary proteins to the plasma membrane. According to this model, functional interactions between signaling proteins would occur only upon receptor activation. However, this simple model does not account for the speed and specificity observed in signal transduction (Riven et al., 2006), and recent evidence suggests the existence of pre-assembled macromolecular signaling complexes containing Kir3 channels built before reaching the cell surface [(Nikolov and Ivanova-Nikolova, 2004; David et al., 2006; Rebois et al., 2006; Riven et al., 2006; Robitaille et al., 2009), reviewed in (Doupnik, 2008; Zylbergold et al., 2010)].

> The notion of Kir3 signaling complexes arose initially from studiesfocusing on the interaction between D2- and D4-dopamine receptors, and Kir3 channels. We showed that the Kir3 channel could be co-immunoprecipitated in HEK 293 cells with either the

D2- or D4- dopamine receptor, and that this interaction required the presence of Gβγ as shown by sequestration of Gβγ by the carboxy-terminal domain of GPCR kinase 2 (GRK2, βARKct; Lavine et al., 2002). We also showed that Kir3 channels interacted with their cognate G proteins well before reaching the cell surface (Rebois et al., 2006). These interactions could be observed with Kir3.1, in the absence of expressed partner subunits, implying that this interaction occured in the endoplasmic reticulum (ER; Robitaille et al., 2009). Using total internal reflection fluorescence (TIRF) microscopy combined with fluorescence resonance energy transfer (FRET), Riven et al. (2006) revealed that complexes of Kir3 and heterotrimeric G proteins exist at rest and that conformational changes in the channel facilitated opening of the channel gate. Furthermore, cell-surface interactions between Kir3, Gβγ, and the δ-opioid receptor (DOR), as detected by bioluminescence resonance energy transfer, have been described, supporting the notion that Kir3 complexes are stable in their journey from biosynthesis to functionality (Richard-Lalonde et al., 2013). Interactions between gamma-aminobutyric acid-B receptors and Kir3 channels have also been observed using an array of techniques (Fowler et al., 2007; Ciruela et al., 2010), with evidence suggesting that these interactions also occur soon after biosynthesis in the ER (David et al., 2006). Data suggesting that such complexes exist *in vivo* was first described by Nikolov and Ivanova-Nikolova (2004) whereby using rat atrial cardiomyocytes, they co-immunoprecipitated G proteins, GRKs, PKA, and protein phosphatases PP1/2, among others, with the KACh channel, indicating the presence of a highly coordinated complex for regulating cardiac excitability. Gβγ has been demonstrated to regulate many effectors [reviewed in (Khan et al., 2013)]. Early signaling complex formation has been observed for other effectors downstream of Gβγ signaling as well. Adenylyl cyclase has also been observed to stably associate with the β2AR (Lavine et al., 2002), with this interaction likely occurring concurrently with biosynthesis of the enzyme (Dupré et al., 2007). The existence of such complexes during receptor and effector biosynthesis along with the fact that Gβγ interacts with multiple signaling partners in the ER suggests that these are organizational events related to the specificity of cellular signaling, and places Gβγ subunits in a good position for acting as a central organizer of signalosome regulation and stability (Dupré et al., 2009; Zylbergold et al., 2010).

The role of Gβγ subunits in channel gating is well established. This article will explore the role of Gβγ dimers during their initial contact with Kir3 channels. We suggest that distinct Gβγ subunits play an important role in orchestrating and fine-tuning parts of the Kir3 channel life cycle.

## **MATERIALS AND METHODS**

#### **REAGENTS**

Blasticidin, hygromycin, Dulbecco's Modified Eagle's Medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin, and tetracycline were from Wisent (St-Bruno, QC, Canada). Lipofectamine 2000 was obtained from Invitrogen (Burlington, ON, Canada). Anti-FLAG M2 affinity agarose beads were from Sigma– Aldrich (St. Louis, MO, USA). Bradford reagent, acrylamide and polyvinylidene fluoride (PVDF) membranes were obtained from

Bio-Rad (Mississauga, ON, Canada). Enhanced chemiluminescence (ECL) Plus reagent was obtained from Perkin Elmer (Woodbridge, ON,Canada). Thefollowing primary antibodies were used: rabbit anti-Kir3.1 (Alomone Labs, Jerusalem, Israel), mouse anti-HA and mouse anti-myc (Covance, Princeton, NJ, USA), rabbit anti-FLAG (Sigma–Aldrich, St. Louis, MO, USA), mouse antiglyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Ambion, Burlington, ON, Canada), rabbit anti-Gβ4 (Santa Cruz, Dallas, TX, USA), mouse anti-Na+/K+-ATPase (Sigma–Aldrich, St. Louis, MO, USA), and mouse anti-β-tubulin (Invitrogen, Burlington, ON, Canada). The rabbit anti-Gβ1 antibody was a generous gift from Dr. Ron Taussig (UT Southwestern Medical Center). The following secondary antibodies were used: goat-anti-rabbit IgG (H+L) conjugated to Alexa488 (Invitrogen, Burlington, ON, Canada), goat-anti-mouse IgG (Fab specific) conjugated to peroxidase and goat-anti-rabbit IgG (whole molecule) conjugated to peroxidase (Sigma–Aldrich, St. Louis, MO, USA). PermaFluor aqueous mounting medium, bovine serum albumin and coverslips were obtained from ThermoScientific (Waltham, MA, USA). siRNAs targeted against Gβ1, Gβ4, Gγ2, Gγ4, Gγ5, and Gγ7 were purchased from Dharmacon (Ottawa, ON, Canada). Primetime qPCR 5 nuclease assays were provided by Integrated DNA Technologies (Coralville, IA, USA). Unless otherwise stated, chemicals were of reagent grade and were purchased from Sigma–Aldrich (St. Louis, MO, USA).

## **CLONING AND GENERATING THE INDUCIBLE CELL LINE**

A previously developed inducible expression system was used to study pulses of Kir3.1 channels as they transit through biosynthetic pathways (Zylbergold et al., 2013). Briefly, extracellularly FLAG-tagged Kir3.1 was subcloned into the pcDNA5-FRT-TO inducible vector using BamHI and NotI restriction sites. The pcDNA5-FRT-TO-FLAG-Kir3.1 (2 μg) was co-transfected with the pOG44 recombinase gene (8 μg) into Flp-InTM T-RexTM 293 parental cells in T75 flasks using Lipofectamine 2000 to generate inducible FLAG-Kir3.1 stables. 48 h post-transfection, cells were passaged and plated into new T75 flasks using fresh media (DMEM supplemented with 5% FBS and 1% penicillin/streptomycin – complete media) containing the selection antibiotics blasticidin (5 μg/mL) and hygromycin (200 μg/mL). Cells were grown to confluency and then used as required for subsequent experiments.

#### **INDUCTION AND TRANSFECTION PROTOCOL**

Inducible FLAG-Kir3.1 stable cells were plated in T75 flasks, allowed to grow for 48–72 h and subsequently transfected with 2 μg of Kir3.2-MYC, FLAG-Gβ1, HA-Gγ2, or various siR-NAs (depending on the experiment) using Lipofectamine 2000 (1 μg:2 μL ratio of DNA:Lipofectamine 2000; 6.25 nM:1 μL siRNA:Lipofectamine 2000, as per manufacturer's protocol) in DMEM (0% FBS, 0% penicillin/streptomycin). Transfection media was then removed and replaced with complete media 5 h later. 24 h post-transfection, cells were induced with 1 μg/mL of tetracycline in DMEM for 30 min at 37◦C, washed three times in DMEM and treated with 5 μg/mL of cycloheximide (CHX) in DMEM for 1 h at 37◦C. Following CHX treatment, cells were washed twice in DMEM, and then incubated at 37◦C in complete

media supplemented with blasticidin for varying periods of time as needed for subsequent experiments.

#### **IMMUNOPRECIPITATION**

Cells were plated in T75 flasks 48–72 h before transfection, cotransfected with 2 μg of Kir3.2-MYC or FLAG-Gβ1, HA-Gγ2, and then induced for Kir3.1-FLAG expression, 24–48 h posttransfection as described above. Transfected cells were washed twice with cold 1X phosphate buffered saline (PBS), harvested in 5 mL of cold 1X PBS, pelleted by centrifugation at 2000 rpm for 8 min at 4◦C, and subsequently resuspended in a buffer containing 5 mM Tris pH 7.4, 2 mM EDTA, with protease inhibitor cocktail (10 μg/mL trypsin inhibitor, 5 μg/mL leupeptin, 50 μg/mL benzamidine). Samples were then subjected to two 10 s bursts with a polytron homogenizer on ice. Debris and unlysed cells were spun down for 5 min at 1000 rpm (4◦C), and the supernatant was then fractionated by a 20 min centrifugation step at 16000 rpm (4◦C) to pellet down the membranes. Pelleted membranes were resuspended in solubilization buffer (75 mM Tris pH 8, 2 mM EDTA, 5 mM MgCl2, 0.5% dodecylmaltoside, protease inhibitor cocktail) and incubated overnight with rotation at 4◦C. The following day, membrane samples were spun for 5 min at 10,000 rpm (4◦C) in order to remove unsolubilized membranes, resuspended in solubilization buffer and then quantified using the Bradford technique (Bio-Rad). In order to immunoprecipitate FLAG-tagged Kir3.1, 500 μg of sample was added to 25 μL of washed anti-FLAG M2 affinity agarose beads and incubated on a rotator overnight at 4◦C. The following day, samples were washed three times [2 min at 1600 rpm (4◦C)] in solubilization buffer supplemented with 300 mM potassium chloride. Bound proteins were eluted off of beads using 60 μL of Tris-buffered saline (TBS) solution (50 mM Tris pH 7.4, 150 mM sodium chloride) containing competitive FLAG peptide (3 μL/100 μL TBS) for 20 min on a 4◦C rotator. Eluted proteins were spun down for 2 min at 1600 rpm (4◦C) and then the supernatant was collected and mixed with 4X sample buffer (62.5 mM Tris, 16.3% glycerol, 2% SDS, 5% β-mercaptoethanol, 0.025% bromophenol blue) in a 1:4 ratio. Samples were then subsequently analyzed by western blot.

## **WESTERN BLOTTING**

For western blotting experiments, 60 μL of samples obtained following immunoprecipitation (IP) and 50 μg of samples from total lysate fractions were loaded on 8% polyacrylamide gels. Gels were electrophoresed for 90 min at 130 V and then transferred to PVDF membranes (Bio-Rad, Mississauga, ON, Canada) for 1 h at 100 V. Following transfer, PVDF membranes were blocked in 5% milk/TBS-containing 1% Tween (TBST) for 1 h at room temperature (RT). Membranes were probed with the primary antibodies as needed [anti-Kir3.1 (1/5000); anti-c-Myc (1/5000); anti-HA (1/5000); anti-Gβ1 (1/5000); anti-Gβ4 (1/400); anti-GAPDH (1/5000); anti-β-tubulin (1/5000); anti-Na+/K+-ATPase (1/5000)] in 5% milk/TBST overnight on a 4◦C rotator. The following day, membranes were washed three times in TBST for 10 min, incubated in the appropriate secondary antibody [antirabbit or anti-mouse HRP (1/20000)] in 5% milk/TBST for 1 h at RT, and then subsequently washed again three times in TBST for

10 min. ECL Plus reagent was then added to the membranes and chemiluminescence was detected using a standard film developer.

#### **IMMUNOFLUORESCENCE**

Cells plated on coverslips in six well plates 48–72 h before transfection were transfected with 300 ng of Kir3.2-MYC or empty vector (pcDNA3), and then induced for Kir3.1-FLAG expression 24–48 h later. At the time of harvest, cells were washed once in 1X PBS pH 7.4 and then fixed in 2% paraformaldehyde (PFA) for 10 min at RT. Cells were then washed three times in 1X PBS pH 7.4 to remove remaining PFA and then blocked in blocking buffer (1X PBS supplemented with 2% BSA) for 1 h at RT. Immediately following blocking, 200 μL blocking buffer with diluted rabbit anti-FLAG antibody (1/250) was applied to each coverslip and incubated overnight at 4◦C. The following day, cells were washed three times in 1X PBS pH 7.4 and incubated with 200 μL blocking buffer supplemented with anti-rabbit Alexa488 (1/500) for 1 h at RT in the dark. Cells were then washed three times in 1X PBS pH 7.4 and coverslips were mounted on glass slides using PermaFluor Aqueous Mounting Medium and then imaged using a Zeiss LSM510 confocal microscope.

#### **REVERSE TRANSCRIPTION AND qPCR**

For assessment of knockdown efficiency of Gγ subunits, total RNA was isolated from siRNA transfected inducible FLAG-Kir3.1 stable cells with TRI reagent using a modified RNA isolation protocol from Ambion. 2 μg of total RNA was reverse transcribed using a Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV-RT, Promega) reaction assay as per the manufacturer's protocol. Reverse transcribed cDNA was subject to qPCR using Custom Primetime qPCR 5- Nuclease assays (IDT) as per the manufacturer's protocol in a Corbett Rotorgene 6000 qPCR instrument. Percentage knockdown efficiencies were calculated using Ctvalues obtained from the qPCR reaction that were subsequently analyzed using the 2−Ct method, using the levels of hypoxanthineguanine phosphoribosyltransferase (HPRT) as the housekeeping gene.

## **RESULTS**

In order to assess the role of Gβγ subunits in the maturation and stability of Kir3.1 channels, we combined our Kir3.1 inducible expression system with RNA interference to knockdown different Gβ and Gγ subunits. Cells co-expressing Kir3.2 and control or different Gβ siRNAs were induced for 30 min with tetracycline and residual leak expression was silenced by a brief treatment with CHX. After washout, Kir3.1 maturation was followed at different time points. The advantage of our system is that it allows for a pulse of channel expression, at physiological protein levels, to mature and traffic to the plasma membrane without saturating the biosynthetic or quality control machinery (Zylbergold et al., 2013). After 6 h, it was observed that sufficient Kir3.1 is available for immunoprecipitation although the immature form is seen immediately after the 30 min pulse (**Figure 1A**; compare times 0 and 6 h in the FLAG IP and Total Lysate blots). The pulse of Kir3.1 expression also indicates that the channel reached maximum levels at 24 h post-induction and then levels subsequently begin to decline. Initial experiments demonstrated the selectivity

**(A)** Flp-In-Trex-FLAG-Kir3.1 cells transfected with myc-Kir3.2 and Gβ1, Gβ4, or control siRNA were induced to express FLAG-Kir3.1 with 1 μg/mL of tetracycline for 30 min and then chased for 0, 6, 24, or 48 h. Upper panel: cells were lysed and FLAG-Kir3.1 was immunoprecipitated using an α-FLAG antibody. Samples were then analyzed by western blot to detect levels of FLAG-Kir3.1 and myc-Kir3.2. Lower panel: total cell lysates showing expression of Kir3.1, Kir3.2, Gβ1, Gβ4, and GAPDH (loading control). Results are representative of three independent experiments. White arrow indicates immature channel proteins while black arrow indicates correctly processed Kir3.1. **(B)** Densitometric analysis of data

significant at *p* < 0.1. **(C)** Flp-In Trex FLAG-Kir3.1 cells were plated on glass coverslips. The cells were transfected with siRNA targeted against Gβ1 and myc-Kir3.2. Cells were subsequently induced to express FLAG-Kir3.1 with 1 μg/mL of tetracycline for 30 min followed by chase times of 2, 7, 24, or 48 h. Cells were fixed with 2% PFA and then labeled (top panels) with rabbit α-FLAG followed by an α-rabbit secondary antibody conjugated to Alexa488 or shown using transmitted light (lower panels). Slides were imaged using a Zeiss LSM510 confocal microscope. Results are representative of a single experiment with several independent fields of cells.

of the siRNAs for Gβ1 and Gβ4 and confirmed efficient silencing of their respective endogenous transcripts in HEK 293 cells as assessed by the depletion of their gene products at the protein level (**Figure 1A**; Total Lysate). Although both Gβ1 and Gβ4 are able to modulate channel function in response to GPCR stimulation (Lei et al., 2000), they are not identical with respect to their effects on channel maturation. In the presence of Kir3.2, Kir3.1 adopts a mature glycosylation phenotype and is trafficked to the cell surface (**Figures 1A–C**). When we knocked down Gβ1, there was little effect on the initial interactions between Kir3.1 and Kir3.2 and early maturational events still occurred. However, the amount of mature and immature channel both decline by 24 h in the absence of Gβ1, suggesting that the holo-channel is less stable and is likely targeted for early degradation. This observation is supported by the fact that much less channel appears on the cell surface when Gβ1 was knocked down, as detected using confocal microscopy (**Figure 1C**). The punctate staining of surface Kir3.1 is consistent with more physiological levels of the channel and again represents one of the advantages of our expression system. The loss of Gβ1 results in a more rapid life cycle for the channel. Knockdown of Gβ1 had no effect on levels of Kir3.2 (**Figure 1A**, Total Lysate), possibly representing either a selective effect on Kir3.1 or a reflection of the fact that bulk expression of Kir3.2 may escape quality control. Future experiments with inducible Kir3.2 constructs may help resolve this issue. Unlike Gβ1, knockdown of Gβ4 had no effect on the stability of the holo-channel, demonstrating a distinct role in channel function that may be restricted to activating the channel in response to receptor stimulation (**Figure 1A**). However, we did observe increased amounts of mature Kir3.1 when Gβ4 was silenced. This may suggest that remaining Gβ1 is now more able to interact with the channel in the absence of a competitor. Our data points to a clear demarcation between the functions of Gβ1 and Gβ4 with respect to Kir3.1 stability and trafficking. Consistent with our RNA interference data, we also noted that overexpression of Gβ1 resulted in holo-channels that were more stable at 48 h post-induction (**Figure 2**). Loss of the Gβ4 subunit also had no effect on levels of Kir3.2 in total cell lysates (**Figure 1A**, Total Lysate) suggesting that their effects are specific for Kir3.1 as well.

Curiously, we noted that knockdown of Gβ4 slightly increased protein expression levels of Gβ1 [also observed in a previous study (Krumins and Gilman, 2006)]. The reason for this is unclear at present but we have preliminary data that suggests it may be through transcriptional effects mediated by Gβ1 (data not shown). This may also reflect the increased channel maturation in the absence of Gβ4, possibly independently of direct competition between the two Gβ subunits. Due to this potential confound of the effect of Gβ4 knockdown on Gβ1 expression, we next performed an experiment to see if a unique effect of Gβ4 could be detected. We knocked down Gβ4 in the presence of siRNA against Gγ2. In the absence of Gγ2, holo-channel stability was also reduced and was not rescued by knockdown of Gβ4 (**Figure 3A**). This makes it likely that Gβ1 and Gγ2 represent the Gβγ pair important for channel maturation. Further, we noted that in the absence of Kir3.2, the stability of the immature forms of Kir3.1 was not affected by knockdown of Gβ4γ2 (**Figure 3B**). This likely indicates that the functional effect of Gβγ on channel stability requires that Kir3.1 be associated with Kir3.2 and that the "stability clock" modulated by Gβγ begins ticking once the channel is set on a pathway of maturation. Additionally, we tested the effect of knockdown of other Gγ subunits on holo-channel stability. **Figure 3** indicates that knockdown of Gγ2 resulted in reduced stability for the Kir3.1/Kir3.2 holo-channel. However, knockdown of Gγ4, Gγ5, or Gγ7 had much more modest effects compared with control siRNA (**Figure 4A**). Knockdown efficiencies of individual Gγ subunits was assessed using RT-qPCR, as endogenous protein expression levels of Gγ isoforms in HEK 293 cells are difficult to visualize using current commercially available anti-Gγ antibodies via western blotting (**Figure 4B**). Changes in expression of the Gγ subunits assessed did not alter expression of Kir3.2

(**Figures 3** and **4**). Finally, overexpression of Gγ2 resulted in holochannels with increased stability (**Figure 5**). Taken together, our results suggest that Gβ1γ2 serve a unique role in channel trafficking that is distinct from their roles in channel activation, which can possibly be subserved by a number of Gβγ dimers with different isoform specificities.

results obtained above. There is a trend in the effects of Gβ1 expression at

48 h, which is reflected in the western blots.

#### **DISCUSSION**

Kir3 trafficking has been a topic of great interest to understand mechanisms mammalian cells use to regulate their excitability. Though much is known regarding Kir3 trafficking as it progresses along the initial stages of its synthesis and maturation, the role

that early interactions with signaling partners such as Gβγ play in channel maturation remain unclear. When expressed alone, recombinant Kir3.1, which contains only one potential site for Nlinked glycosylation, migrates as a doublet with a molecular mass of 54 and 56 kDa, with the upper band being the core-glycosylated, immature form of the protein (Krapivinsky et al., 1995a; Kennedy et al., 1999). Upon treatment with either endoglycosidase H, an

enzyme that selectively removes N-linked glycosyl moieties from proteins that have not been processed in the Golgi, or endoglycosidase F, an enzyme that non-selectively removes all N-linked sugar residues, the 56 kDa band is virtually abolished, confirming its residence in the ER (Kennedy et al., 1999). Co-expression of Kir3.4 with Kir3.1, results in a fully mature Kir3.1 subunit capable of being properly glycosylated (Kennedy et al., 1999). The role

**mature Kir3.1 stability. (A)** Flp-In Trex FLAG-Kir3.1 cells were transfected with Gγ2, 4, 5, or 7 targeted siRNA and myc-Kir3.2. The cells were subsequently induced to express FLAG-Kir3.1 with 1 μg/mL of tetracycline for 30 min followed by 5 or 48 h chase times. Cells were lysed and FLAG-Kir3.1 was immunoprecipitated as described above. Upper panel: samples were analyzed by western blot to detect levels of FLAG-Kir3.1 and myc-Kir3.2. Lower panel: total cell lysates showing expression of Kir3.1, Kir3.2, and β-tubulin (as a loading control). Results are shown from a single experiment. White arrow

correctly processed Kir3.1. **(B)** Flp-In Trex FLAG-Kir3.1 cells were transfected with 50 nM of specific siRNA targeted against Gγ2, Gγ4, Gγ5, or Gγ7. Cells were then lysed in TRI reagent 72 h post-transfection, total RNA was isolated, and the extent of Gγ knockdown was assessed by RT-qPCR using probe specific qPCR assays. Data is depicted as mean ± S.E.M. and is representative of *n* = 3 experiments. Statistical significance was evaluated using an unpaired Student's *t*-test where \* indicates *p* < 0.05 and \*\* indicates *p* < 0.01.

of Kir3.1 glycosylation may be one of quality control, as Kir3.1 subunits containing a mutation preventing glycosylation can still interact with partner subunits and traffic to the plasma membrane yielding typical current amplitudes (Pabon et al., 2000).

There is a trend in the effects of Gγ2 expression at 48 h, which is reflected

Here we show that a number of maturational steps for Kir3.1 are strictly dependent on the presence of Kir3.2. In the absence of Kir3.2, Kir3.1 subunits remain immature and the loss of Gβγ subunits does not affect this outcome. Though most studies have focused on the role of Kir3.4 in the trafficking of Kir3 channels, Kir3.2 subunits have been found to play a similar vital role in

forming functional Kir3.1-containing channels at the cell surface. Kir3.2 subunits are able to form both homotetramers as well as heterotetramers with either Kir3.1 or Kir3.3 which can then be targeted to the plasma membrane (Jelacic et al., 2000). Using deletion analysis, it was suggested that an ER export signal found at both the N- and C- terminus of Kir3.2 is vital for the cell surface delivery of Kir3.1/3.2 heterotetramers (Ma et al.,2002). Though no well-defined ER export motif has been characterized for Kir3.3, it has been shown to form tetrameric channels with both Kir3.1 and Kir3.2 subunits (Jelacic et al., 1999). Altogether, Kir3 trafficking from the ER appears to be a highly regulated process, with distinct combinations being available at the plasma membrane or targeted for degradation depending on the needs of the cell. Interestingly, when we inhibit the proteasome with MG-132, Kir3.1 can traffic to the cell surface (P.Z. unpublished observations), demonstrating a likely role for ER-associated degradation (ERAD) in the quality control of unpartnered Kir3.1 subunits.

Much attention has been paid to the specificity of Gβγ dimers in the activation of Kir3 channels. To date, Gβ5 stands out as an outlier in the Gβ family in that Gβ5-Gγ combinations significantly downregulate channel activation, leading to the suggestion that it acts as a competitive antagonist for Kir3 activation by other Gβγ subunit combinations (Lei et al., 2000, 2003). Interestingly, other Gβγ combinations show similar abilities to activate channels. Some Gβγ selectivityfor Kir3.2 has been noted, in that Gβ subtypes 1–3 preferentially interact with this subunit (Robitaille et al.,2009). Most studies have found that any of the four Gβ subunits could be expressed with distantly related Gγ subunits (i.e., Gγ2 vs. Gγ11) and all combinations could bind and activate Kir3 channels with similar efficacy (Lei et al., 2000).

Gβ1γ2, apart from its role in channel opening shared with other Gβγ dimers, seems to play a unique role in preserving maturing channels from degradation as they transit to the cell surface (summarized in **Figure 6**). Our data indicate that Gβ1γ2 prolongs the lifetime of the Kir3.1/Kir3.2 heterotetramer, although levels of Kir3.2 are not altered *per se*. This may suggest that Kir3.2 homotetramers might be differentially regulated by other factors although under conditions of bulk overexpression, it is difficult to prove this. Placing the other Kir3 isoforms under the control of inducible promoters using our label-free pulse-chase technique will help resolve these issues. It is clear that the roles Gβγ play in Kir3 activity and regulation are quite complex and likely exceed our current understanding. There are numerous potential Gβγ interacting domains on the tetrameric channel and it is evident that not all of these simply underlie channel activation and gating (i.e., Gβγ-associated channels need not be active until they reach the membrane). Gβγ subunits are involved in channel synthesis, trafficking, plasma membrane stability and degradation (Robitaille et al., 2009; Zylbergold et al., 2010), and it is likely that novel roles beyond mere channel activation will be uncovered in the years to come. Here, we have described some approaches that will allow these issues to be resolved with greater clarity.

#### **ACKNOWLEDGMENTS**

This study was supported by a Canadian Institutes of Health Research (CIHR) grant to Terence E. Hébert (CIHR; MOP-36379). Terence E. Hébert was a "Chercheur National" of the "Fonds de la

in the western blots.

**FIGURE 6 | Distinct Gβγ effects on Kir3 stability.** Under normal conditions, pulse induction of Kir3.1 expression in the presence of Kir3.2 forms heterotetrameric channels of mature Kir3.1 and Kir3.2 (light green), which can traffic to the cell surface and remain expressed at the plasma membrane for up 48 h. siRNA mediated knockdown of Gβ1 or Gγ2 has negligible effects on immature Kir3.1 channels (dark green) but impairs Kir3.1 maturation and hastens the loss of channel at the cell surface suggesting a stabilizing roles for the Gβ1γ2 dimer. These effects of Gβ1γ2

knockdown can be mediated through reduced anterograde trafficking (dashed black arrow) and/or enhanced channel internalization (solid blue arrow). Regardless of the Gβγ pair associated with Kir3 channels in the ER, these are trafficked out to the cell surface. However, those associated with Gβ1γ2 seem to persist longer than those associated with other Gβγ combinations. This suggests that additional functions exist for some Gβγ combinations, independent of their ability to open the channel in response to GPCR stimulation.

Recherche en Santé du Québec" (FRSQ) from 2008–2012. Peter Zylbergold, Rory Sleno, and Shahriar M. Khan were all supported by scholarships from the McGill CIHR Drug Development Training Program. Mark A. Belhke and Ashley M. Jacobi are employed by Integrated DNA Technologies, Inc. (IDT) which offers oligonucleotides for sale similar to some of the compounds described in the manuscript. IDT is however not a publicly traded company and these authors personally do not own any shares/equity in IDT.

## **REFERENCES**

Kir 3 inwardly rectifying potassium channels. *Cell. Signal.* 18, 2172–2181. doi: 10.1016/j.cellsig.2006.05.014

activity through interactions with the βγ subunits of G proteins. *J. Biol. Chem.* 277, 6088–6096. doi: 10.1074/jbc.M104851200

**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.

*Received: 11 February 2014; accepted: 25 March 2014; published online: 14 April 2014. Citation: Zylbergold P, Sleno R, Khan SM, Jacobi AM, Belhke MA and Hébert TE (2014) Kir3 channel ontogeny – the role of G*βγ *subunits in channel assembly and trafficking. Front. Cell. Neurosci. 8:108. doi: 10.3389/fncel.2014.00108*

*This article was submitted to the journal Frontiers in Cellular Neuroscience.*

*Copyright © 2014 Zylbergold, Sleno, Khan, Jacobi, Belhke and Hébert. This is an openaccess 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) or licensor 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.*

## Kir3 channel signaling complexes: focus on opioid receptor signaling

## *Karim Nagi 1,2 and Graciela Pineyro1,2,3\**

*1 Département de Pharmacologie, Faculté de Médecine, Université de Montréal, Montreal, QC, Canada*

*2 Centre de Recherche du CHU Sainte-Justine, Montréal, QC, Canada*

*3 Département de Psychiatrie, Faculté de Médecine, Université de Montréal, Montréal, QC, Canada*

#### *Edited by:*

*Leigh Anne Swayne, University of Victoria, Canada*

#### *Reviewed by:*

*Lin Tian, University of California, Davis, USA Nathan Dascal, Tel Aviv University, Israel*

#### *\*Correspondence:*

*Graciela Pineyro, Department of Psychiatry, Centre de Recherche du CHU Ste-Justine, University of Montreal, 3175, Côte Ste-Catherine, Bureau 2722, Montréal, QC H3T 1C5, Canada e-mail: graciela.pineyro.filpo@ umontreal.ca*

Opioids are among the most effective drugs to treat severe pain. They produce their analgesic actions by specifically activating opioid receptors located along the pain perception pathway where they inhibit the flow of nociceptive information. This inhibition is partly accomplished by activation of hyperpolarizing G protein-coupled inwardly-rectifying potassium (GIRK or Kir3) channels. Kir3 channels control cellular excitability in the central nervous system and in the heart and, because of their ubiquitous distribution, they mediate the effects of a large range of hormones and neurotransmitters which, upon activation of corresponding G protein-coupled receptors (GPCRs) lead to channel opening. Here we analyze GPCR signaling via these effectors in reference to precoupling and collision models. Existing knowledge on signaling bias is discussed in relation to these models as a means of developing strategies to produce novel opioid analgesics with an improved side effects profile.

**Keywords: GIRK, opioid, signaling, analgesia, bias, signaling complexes, functional selectivity, allosteric modulation**

Opioids produce their actions via the activation of μ (MOR), δ (DOR), and κ (KOR) receptors (Pert and Snyder, 1973; Simon et al., 1973) all of which couple to heterotrimeric Gi/o proteins. As such they share adenylyl cyclase, Phospholipase Cβ [PLCβ, N-type Ca2+ (Cav2.2)] channels and G-protein coupled inward rectifier K+ channels (GIRKs or Kir3 channels) as canonical Gα and Gβγ effectors. The two channel effectors prominently mediate analgesic effects of opioids. For example, studies in rodents have shown that activation of MORs on small, unmyelinated nociceptors (Scherrer et al., 2009; Heinke et al., 2011; Bardoni et al., 2014) contribute to the analgesic effects of morphine (Wei et al., 1996; Andrade et al., 2010), and this is done via inhibition of N-type Ca+2 channels that control neurotransmitter release from these primary afferents (Glaum et al., 1994; Wrigley et al., 2010). Kir3 channel contribution to opioid analgesia has also been demonstrated in animal models (Ikeda et al., 2000; Mitrovic et al., 2003; Marker et al., 2005), and their relevance in the clinical response to opioid analgesics has been further confirmed by

**Abbreviations:** GIRK, G protein activated inward rectifier K+; GPCRs, G protein-coupled receptors; DORs, Delta opioid receptors; MORs, Mu opioid receptors; KORs, Kappa opioid receptors; PLC, Phospholipase C; GABA, Gamma-aminobutyric acid; DRG, dorsal root ganglion; DAMGO, [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin; KCNJ6, Inwardly-rectifying potassium channel, subfamily J, member 6; KCNJ3, Inwardly-rectifying potassium channel, subfamily J, member 3; 5-HT, 5-hydroxytryptamine; MAPK, Mitogen-activated protein kinases; βarr, βarrestin; cAMP, Cyclic adenosine monophosphate; VTA, Ventral tegmental area; PIP2, Phosphatidylinositol 4,5-bisphosphate; GDP, Guanosine diphosphate; GTP, Guanosine triphosphate; PSD95, Postsynaptic density protein 95; SAP97, Synapse-associated protein 97; FRET, Fluorescence resonance energy transfer; BRET, Bioluminescence resonance energy transfer; PC12, Neuron-like pheochromocytoma.

gene linkage analyses (Nishizawa et al., 2009; Lotsch et al., 2010; Bruehl et al., 2013). In addition, the use of genetically modified mice lacking different channel subunits has further established activation of Kir3 channels as a pervasive analgesic mechanism which, in addition to opioids, also mediates pain modulation by α2 adrenergic, muscarinic, GABAB and cannabinoid receptor ligands (Blednov et al., 2003; Mitrovic et al., 2003). From this perspective the development of direct Kir3 channel activators may be considered a promising strategy for the development of novel analgesics. However, enthusiasm for this particular approach has remained limited given the possibility of widespread side-effects associated with administration of direct channel openers (Lujan et al., 2014). Indeed, these would result from the great variety of physiological responses mediated by Kir3 channels, including heart frequency control (Wickman et al., 1998; Bettahi et al., 2002), memory (Wickman et al., 2000), learning (Wickman et al., 2000; Cooper et al., 2012), as well as their participation in different pathological conditions including development of seizures (Signorini et al., 1997), generation of anxiety states (Blednov et al., 2001; Pravetoni and Wickman, 2008) and contribution to abnormal plasticity by drugs of abuse (Padgett et al., 2012; Hearing et al., 2013). Consequently, the prevailing strategy for engaging Kir3-mediated analgesia has remained the activation of pain-modulating GPCRs (McAllister et al., 1999; Lujan et al., 2014). Among them MORs, DORs and KORs all three mediate effective analgesia but display different side effects that distinctively limit their therapeutic use (Kieffer and Gaveriaux-Ruff, 2002; Bruchas and Chavkin, 2010; Gaveriaux-Ruff and Kieffer, 2011). Here we will summarize Kir3 channel contribution to desired and undesired responses of opioid analgesics and address the question of whether biasing opioid signaling toward these effectors could help develop better opioid analgesics with a reduced side effects profile.

## **Kir3 CHANNEL SIGNALING AND OPIOID-DEPENDENT ANALGESIA**

Kir3 channels are formed by tetrameric association of four different subunits (Kir3.1-3.4). Although all four of them are expressed in peripheral (Gao et al., 2007) and central nervous system (Wickman et al., 2000; Saenz Del Burgo et al., 2008; Fernandez-Alacid et al., 2011), neural channels are most frequently formed by association of Kir3.1, 3.2 and 3.3 subunits (Liao et al., 1996; Hibino et al., 2010; Luscher and Slesinger, 2010). The subunit combination that forms the channel will depend on the neuronal population involved (Inanobe et al., 1999; Cruz et al., 2004) and the cellular compartment (Koyrakh et al., 2005; Fernandez-Alacid et al., 2009) in which channels are found. Nonetheless, Kir3.1/3.2 heterotetramers are considered prototypical neuronal channels.

Kir3 channel contribution to opioid analgesia was initially suggested by the fact that mice carrying a missense mutation which renders Kir3.2 subunits insensitive to G protein activation (Patil et al., 1995; Navarro et al., 1996) displayed reduced analgesic responses to morphine (Ikeda et al., 2000, 2002). These observations were subsequently confirmed using null mice for different subunits (Mitrovic et al., 2003; Marker et al., 2005), which allowed to also implicate Kir3.1 (Marker et al., 2004) and Kir3.3 (Marker et al., 2002) in pain modulation. Knock-out of Kir3.2 and 3.3 was shown to interfere with morphine's ability to prolong avoidance behavior in the hot plate test. Since this response involves supraspinal integration, it can be concluded that both subunits contribute to mechanisms of opioid analgesia at this level (Marker et al., 2002; Mitrovic et al., 2003). Possible sites of supraspinal Kir3-mediated analgesia may include thalamus and limbic cortex, both of which express Kir3.1, 3.2 and 3.3 subunits (Saenz Del Burgo et al., 2008; Fernandez-Alacid et al., 2011) as well as opioid receptors (Le Merrer et al., 2009). The midbrain periaqueductal gray seems less likely since in this nucleus opioid actions are primarily presynaptic reducing neurotransmitter release via a mechanism that involves phospholipase A2, arachidonic acid and 12-lipoxygenase, which leads to modulation of voltage-dependent potassium channels (Vaughan and Christie, 1997; Vaughan et al., 1997).

Genetically engineered mice lacking Kir3.1 or Ki3.2 subunits also display reduced responses to intrathecal administration of morphine in the tail flick test (Marker et al., 2004, 2005) implicating both subunits in spinal mechanisms of opioid analgesia. This interpretation is supported by reports locating Kir3.1 and Kir3.2 subunits to lamina II interneurons that co-express μ opioid receptors (Marker et al., 2006). Functional studies also indicated that silencing of Kir3.1 or Kir3.2 subunits, or intrathecal infusion of Kir3 channel blocker tertiapin-Q interfered with the analgesic response by MOR and DOR agonists administered by the same route (Marker et al., 2005). In contrast, Kir3.3 ablation was without effect on the analgesic response elicited by intrathecal morphine (Marker et al., 2004), arguing against its significant contribution at this level. The latter observation is also in

keeping with immunohistological studies which reported absence of Kir3.3 labeling in the dorsal horn (Marker et al., 2004).

In addition to their participation in acute opioid analgesia, spinal Kir3 channels seem to mediate neuroadaptations that modulate responsiveness to opioids in conditions such as inflammatory or cancer-related pain. This possibility is particularly suggested by reports indicating that carrageenan-inflammation (Gonzalez-Rodriguez et al., 2010) and bone cancer (Gonzalez-Rodriguez et al., 2012) enhance the inhibitory effect of Kir3 channel blocker tertiapin-Q on analgesia induced by intrathecal administration of morphine.

Apart from brain and spinal cord, opioid receptors are also present in sensory neurons of the dorsal root ganglion (DRG) (Li et al., 1998; Gendron et al., 2006; Wu et al., 2008; Wang et al., 2010) and are transported to peripheral terminals where they mediate analgesic actions of peripherally injected opioids (Hassan et al., 1993; Obara et al., 2009; Vadivelu et al., 2011). Transcripts for Kir3.1/3.2 subunits have been detected in human DRGs and in rat nociceptors, but not in mice (Gao et al., 2007; Nockemann et al., 2013). This distribution parallels differences in species sensitivity to peripheral administration of opioids suggesting that when expressed, DRG Kir3 subunits actively participate in opioid analgesia.  Moreover, the active contribution of Kir3.2 channels to peripheral opioid analgesia has now been experimentally established using transgenic mice genetically engineered to express these subunits in sensory neurons. Unlike their wild-type counterparts, the latter display an analgesic response to plantar application of MOR agonist DAMGO (Nockemann et al., 2013).

It is important to bear in mind that most of the evidence analyzed thus far links Kir3 channel function to opioid analgesia in animal models. However, the latter have shown somewhat limited success in identifying and validating analgesic targets of clinical relevance (Mogil, 2009). Hence, from this perspective, any evidence linking Kir3 channels to therapeutic response in humans is of specific interest. At least three studies have now shown that variations in the gene coding for Kir3.2 subunits (*KCNJ6*) influence opioid dose requirements for both acute management of postoperative pain (Nishizawa et al., 2009; Bruehl et al., 2013) and for pain control in chronic patients (Lotsch et al., 2010). Similar assessment of Kir3.1 (*KCNJ3*) variations showed no effect (Bruehl et al., 2013).

## **OPIOID SIDE EFFECTS AND Kir3 CHANNEL SIGNALING**

In spite of the established analgesic efficacy of MOR, DOR, and KOR agonists, activation of the different receptor subtypes results in a distinct set of side effects which limit therapeutic application of their agonists. Constipation, nausea, respiratory depression, tolerance, dependence, and abuse are among the most common undesired effects of clinically available MOR agonists (Ballantyne and Shin, 2008; Morgan and Christie, 2011). Many of these undesired actions, particularly respiratory depression (Cheng et al., 1993; Gallantine and Meert, 2005), gastrointestinal side effects (Tavani et al., 1990; Gallantine and Meert, 2005; Feng et al., 2006) and physical dependence (Cowan et al., 1988; Codd et al., 2009) are less severe or absent with DOR agonists. Moreover, although DORs and MORs are both involved in reward response to physiological stimuli (Charbogne et al., 2014), DORs are not directly involved in assigning reward value to stimuli but instead facilitate predictive learning and influence the choice of action in face of contingencies that involve reward (Laurent et al., 2012). In keeping with this functional profile, DOR agonists do not facilitate intracranial self-stimulation (Do Carmo et al., 2009), are not discriminated as morphine substitutes (Gallantine and Meert, 2005) and do not display reinforcing properties in non-human primates (Banks et al., 2011). Nonetheless, despite these advantages, potential for tolerance (Pradhan et al., 2010; Audet et al., 2012) remains a limitation for long-term use of DOR agonists, as does their propensity to produce hippocampal hyperexcitability that may lead to seizures. The latter are produced both by DOR (De Sarro et al., 1992; Broom et al., 2002; Jutkiewicz et al., 2005) and MOR agonists (Drake et al., 2007). In the specific case of DOR agonists, hyperexcitability is brief and non-lethal. Moreover, seizures are rare at analgesic doses (Jutkiewicz et al., 2006), they do not appear with all ligands (Le Bourdonnec et al., 2008; Saitoh et al., 2011) and can be suppressed by controlling the rate of agonist administration (Jutkiewicz et al., 2005). Thus, overall, side effects of DORs agonists are considerably milder as compared to those of MORs, and their proven efficacy in chronic pain management make DORs especially attractive as targets for the development of novel analgesics (Gaveriaux-Ruff and Kieffer, 2011). This notion is further reinforced by the fact that DOR agonists display antidepressant properties (Chu Sin Chung and Kieffer, 2013) which could be of additional benefit in controlling negative affect, frequently associated with prolonged pain syndromes (Goldenberg, 2010). Unlike DOR agonists, KOR-mediated analgesia is typically associated with stress, depression, and dysphoria (Bruchas and Chavkin, 2010; Van't Veer and Carlezon, 2013) which, together with their tendency to induce tolerance (McLaughlin et al., 2004; Xu et al., 2004), would make these ligands less attractive for the treatment of chronic pain.

Kir3 channel function has been associated with some of the undesired effects of opioid analgesics. For example, reduced GABAergic activity in hippocampal dentate gyrus increases the excitability of glutamatergic granule cells and may reduce the threshold for seizures (Drake et al., 2007). MORs and DORs agonists silence hippocampal GABAergic interneurons by a mechanism that involves both Kir3 (Luscher et al., 1997) and voltage-dependent K+ channels (Wimpey and Chavkin, 1991; Moore et al., 1994), explaining their documented tendency to produce seizures (Drake et al., 2007). MORs agonists also enhance excitability and firing activity of dopaminergic neurons of the ventral tegmental area (Gysling and Wang, 1983), and do so by hyperpolarization of local interneurons (Johnson and North, 1992; Bonci and Williams, 1997). Like in hippocampus, disinhibition is mediated through Kir3 channel activation (Luscher et al., 1997) and its consequence is the enhanced release of dopamine in corticolimbic areas which is thought to facilitate compulsive behaviors characteristic of addiction (Luscher and Ungless, 2006). Finally, Kir3 channel activation by KORs modulates firing activity of dorsal raphe 5-HT neurons (Bruchas et al., 2007; Lemos et al., 2012). Dynorphin release during repeated stress exposure produces sustained activation of these receptors driving p38α-MAPK activity and subsequent Kir3.1 subunit phosphorylation (Lemos et al., 2012). As a consequence 5-HT neuron firing activity becomes deregulated, possibly contributing to dysphoric effects of uncontrollable stress and to aversive actions of KOR agonists (Bruchas et al., 2007).

Additional undesired actions of opioids analgesics involve signaling effectors other than Kir3 channels. For example, constipation and respiratory depression have been associated with βarrestin (βarr)-mediated signaling by MOR agonists (Raehal et al., 2005; Dewire et al., 2013). The use of βarr-knockout mice has also suggested that DOR agonist tendency to induce seizures may involve this regulatory protein, and biased agonists that fail to recruit βarrs are being developed as a means of further improving the side effects profile of analgesics acting at this receptor subtype. Kir3-independent side effects also include compensatory changes in the cyclase pathway (cyclase superactivation). These are triggered by sustained inhibition of cAMP production (Christie, 2008) and have been shown to contribute to physical dependence (Han et al., 2006; Cao et al., 2010; Yang et al., 2014) and analgesic tolerance of opioid agonists (Javed et al., 2004; He and Whistler, 2007; Bobeck et al., 2014).

Data summarized thus far indicate that undesired actions of opioid ligands segregate according to receptor subtype, and within each subtype, desired and unwanted effects are not all necessarily mediated by the same effectors. Thus, undesired actions of DOR and KOR-activating analgesics are less than those of MOR ligands, and among the former, the presence of antidepressant properties and lack of dysphoric actions makes DOR specifically interesting as putative targets for the management of chronic pain syndromes (Gaveriaux-Ruff and Kieffer, 2011; Pradhan et al., 2011). Tolerance however, remains a drawback that limits further development of DOR-acting analgesics. This limitation could be, at least, partly addressed by taking advantage of biased signaling since effectors that mediate cellular tolerance and analgesia seem partly segregated. Indeed, as detailed above, while analgesic actions are generally mediated via modulation of channel effectors, adaptations of the cAMP cascade seem to account for at least some of the manifestations of tolerance (Javed et al., 2004; He and Whistler, 2007; Bobeck et al., 2014). Based on these observations, opioid ligands that specifically bias their pharmacological stimulus toward channel effectors could conceivably conserve analgesic properties while displaying reduced potential for tolerance, particularly the component that depends on cyclase adaptations. In the following sections, we will consider possible strategies to direct opioid signaling toward Kir3 channels. However, before doing so it is worth revising the events that lead to channel activation.

#### **Kir3 CHANNEL ACTIVATION VIA GPCRS**