 TRIC-B1. The anomalous peaks of Rb+ contoured at 3.2σ are shown as blue meshes. The data were collected at 0.8147 Å wavelength and the map resolution is 3.7 Å. d, The binding site of Ca2+ on TRIC-B1. The magenta anomalous peaks are contoured at 3σ. The data were collected at 3.0 Å wavelength and the map resolution is 4.0 Å. Besides the one on Ca1 site, the other magenta peaks within the boxed area in the transmembrane region are contributed by the sulfur atoms from Cys or Met residues and phosphorus atoms from the PIP2 molecules. e, Verification of the Ca2+-binding site with its surrogate ion Ba2+. The anomalous peak of Ba2+ is contoured at 5σ and shown as grey meshes. The data was collected at 1.4 Å wavelength and the map resolution is 4.2 Å. The protein backbones of the three monomers are shown as ribbon models in purple, lime and orange, respectively. The PIP2 molecules are shown as stick models. Stereo pairs are shown in all panels, and the views are from luminal side along C3 axis (a), or side views along the membrane plane (b–e).
Extended Data Figure 4 Comparing the structure of C. elegans TRIC-B1 monomer with the canonical tetrameric K+ channel structure (KcsA).
a, The overall structure of TRIC-B1 monomer with the pore-lining key residues and PIP2 molecule shown as stick models. The purple sphere is a predicted K+ ion binding site according to the position of Rb+ peak. b, A zoom-in view of the pore centre in TRIC-B1 channel with a binding site selective for monovalent cation. c, The overall structure of the KcsA channel with two subunits shown. PDB code: 1BL8. The K+ ions and water molecules are shown as purple and red spheres, respectively. d, A zoom-in view of the selectivity filter of KcsA channel. It is mainly contributed by the backbone carbonyl groups.
Extended Data Figure 5 Electrophysiological characterization of the activities of C. elegans TRIC-B1 channel.
a, Representative single channel currents recorded at ± 40 mV (in symmetrical 210 mM KCl solutions) through the inside-out patch-clamp electrophysiology using the reconstituted GUVs. b, The normalized all-points amplitude histogram analysis of the data with three major open states (+40 mV). For the plot, the bin width is set at 0.03 pA/bin and the total count of events is normalized to 1.0. The distribution data were fit by a sum of 4 Gaussians, with means of 0, 1.81, 4.00, 6.43 pA, representing the closed, 1st, 2nd and 3rd open levels labelled as C and O1–O3. c, The current–voltage (I/V) relationships of TRIC-B1 channel in O1–O3 states. The error bars represent the s.e.m. Numbers of measurements are labelled nearby the data point symbols (n = 3, 4 or 5). d, The probabilities of TRIC-B1 channel being open or closed compared to the estimated probabilities based on the binomial model. P(k)exp is measured experimentally basing on the counts of events, while P(k)binomial represents the binomial distribution probability of k independent channels being open simultaneously. The P(k)exp/P(k)binomial ratios, corresponding to the closed (C), one channel open (O1), two channel open (O2) and three channel open (O3) states, are presented in the graph. The data were derived from amplitude histogram analysis on single-channel data recorded at +40 mV. e, A table listing the probability data based on experimental observation (P(k)exp) and the binomial model (P(k)binomial). f, Mean open dwell time analyses on the data recorded from patches containing one (n = 22 from 3 patches) and three (n = 4 from 3 patches) wild-type channels. g, Dwell time analyses on the open states of the data recorded from patches containing one (n = 6 from 3 patches) and three (n = 7 from 1 patch) K129A/R133L mutant channels. The demo traces of wild-type and K129A/R133L mutant are shown in Fig. 3e, f. For kinetic analyses on the dwell time distributions, the data set were fit with two components, namely τ1 (left) and τ2 (right) by using the log probability exponential function. The s.e.m. of data from different patches is indicated by the error bars shown in f and g.
Extended Data Figure 6 Cysteine accessibility of the pore-lining residues within C. elegans TRIC-B1 channel.
a, Top view of a TRIC-B1 monomer from cytosolic side. The view shows that the cytoplasmic vestibule of the pore (orange elliptical ring) is mainly shaped by M1 (purple), M4 (green) and M5 (M5a and M5b, blue). b–d, The positions of Met38 (b), Ala126 (c) and Ser166 (d) on the pore-lining M1, M4 and M5 helices, respectively. The sphere models highlight these three pore-lining residues selected for mutation into cysteine and subject to cysteine-accessibility tests by using MTSET during electrophysiological studies. e, Single-channel recording for the three cysteine mutants of TRIC-B1 measured at ±40 mV. ‘Cys-less’ indicates the template (with all endogenous Cys replaced with Ser) used for introducing the M38C, A126C or S166C mutation, and it is included as a control. The measurements were performed under standard conditions without MTSET (dark traces on the left, control) followed by addition of 2 mM MTSET in the bath solution (red traces recorded with patches being incubated with MTSET for 5–6 min).
Extended Data Figure 7 The oligomeric state analyses and fluorescence-based K+ flux assays on the wild-type C. elegans TRIC-B1 and mutants.
a, Chemical cross-linking patterns of purified wild-type and K129A/R133L protein samples. The cross-linker is 0–10 mM glutaraldehyde (GA). The reaction was performed at 25 °C for 20 min. Note the markedly reduced intensities of dimeric and trimeric bands in K129A/R133L sample compared to the wild-type sample. b, Gel filtration profiles of the wild-type and K129A/R133L mutant of TRIC-B1 protein on a Superdex 200 10/300 GL column. The arrows indicate the major peak fractions of K129A/R133L mutant and wild-type proteins. Note the right shift of the mutant peak compared to that of the wild type. c, western blot of the wild-type TRIC-B1 and K129A/R133L mutant proteins being cross-linked on the membrane. The cross-linker used is 0–10 mM disuccinimidyl suberate (DSS, a membrane-permeable cross-linker), and the cross-linking reactions were performed at 25 °C for 30 min. Note the reduced fractions of dimeric and trimeric bands in K129A/R133L sample compared to the wild-type sample. d, The K+ flux activity of wild-type TRIC-B1 in response to different [Ca2+] inside the liposome. No Ca2+ was added in the external buffer. e, The effect of titrating Ca2+ in the external buffer on the wild-type TRIC-B1 activity. The internal solution of the proteoliposome sample is constant with 5 mM Ca2+. f, The activities of W180A mutants measured under the conditions with internal [Ca2+] varied at 0 or 5 mM while no Ca2+ was added in the external buffer as in d. The blue arrows indicate the points of adding the proton ionophore (CCCP), while the red asterisks indicate the point when the K+ ionophore valinomycin was added. g, The competitive inhibition model fitting for the estimation of IC50 value of external [Ca2+] versus the relative activity of the wild-type TRIC-B1. The relative activity is derived from the declined value of the normalized fluorescence at 450 s reaction time. Error bars are s.e.m., n = 4.
Extended Data Figure 8 The cytoplasmic Ca2+-binding sites and conformational change of the M5–6 loop region induced by Ca2+ binding.
a, Electrostatic surface presentations of TRIC-B2 trimer (without Ca2+ bound) and TRIC-B1 trimer (with Ca2+ bound). The colour codes for the electrostatic potential on the surfaces of the TRIC-B1/B2 trimers are: red, −15 kTe−1; white, neutral; blue, +15 kTe−1. The Ca2+ ion bound to TRIC-B1 trimer is presented as a green sphere. The green arrows indicate the regions around the trimeric centres involved in binding Ca2+. The magenta stars label the pore region within each monomer. b, 2Fo − Fc density of Ca2+ ion and putative water molecules surrounding it. The number labels nearby the dash lines indicate the distances (Å) between water and the side chain