Pore architecture of TRIC channels and insights into their gating mechanism

Abstract

Intracellular Ca2+ signalling processes are fundamental to muscle contraction, neurotransmitter release, cell growth and apoptosis1,2. Release of Ca2+ from the intracellular stores is supported by a series of ion channels in sarcoplasmic or endoplasmic reticulum (SR/ER)3,4. Among them, two isoforms of the trimeric intracellular cation (TRIC) channel family, named TRIC-A and TRIC-B, modulate the release of Ca2+ through the ryanodine receptor or inositol triphosphate receptor, and maintain the homeostasis of ions within SR/ER lumen5,6. Genetic ablations or mutations of TRIC channels are associated with hypertension, heart disease, respiratory defects and brittle bone disease7,8,9,10,11,12. Despite the pivotal function of TRIC channels in Ca2+ signalling5,13,14, their pore architectures and gating mechanisms remain unknown. Here we present the structures of TRIC-B1 and TRIC-B2 channels from Caenorhabditis elegans in complex with endogenous phosphatidylinositol-4,5-biphosphate (PtdIns(4,5)P2, also known as PIP2) lipid molecules. The TRIC-B1/B2 proteins and PIP2 assemble into a symmetrical homotrimeric complex. Each monomer contains an hourglass-shaped hydrophilic pore contained within a seven-transmembrane-helix domain. Structural and functional analyses unravel the central role of PIP2 in stabilizing the cytoplasmic gate of the ion permeation pathway and reveal a marked Ca2+-induced conformational change in a cytoplasmic loop above the gate. A mechanistic model has been proposed to account for the complex gating mechanism of TRIC channels.

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Figure 1: The overall structure of C. elegans TRIC-B1 channel.
Figure 2: The ion permeation pathways of C. elegans TRIC-B channels.
Figure 3: Structural and functional roles of PIP2 in C. elegans TRIC-B1.
Figure 4: The gating mechanism of TRIC-B channels.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Coordinates and structure factors have been deposited in the Protein Data Bank under accession codes 5EGI (C. elegans TRIC-B1) and 5EIK (C. elegans TRIC-B2).

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Acknowledgements

We thank J. Y. Sun for discussions on electrophysiology and sharing electrophysiological devices, F. Q. Yang for advice on mass spectrometry analysis of lipid samples, Y. H. Huang and X. C. Zhang for discussions on structure and mechanism, X. Y. Liu and X. B. Liang for technical assistance with biochemistry, crystal handling and data collection, Y. Han at the IBP core facility for the support during in-house X-ray data collection, the staff at SSRF and PF for their support during synchrotron data collection. The experiments at PF were performed under the approval of the Photon Factory Program Advisory Committee (Proposal No: 2014G179). This project is financially supported by the National 973 Program from Chinese Ministry of Science and Technology (Grant 2014CB910301), the Strategic Priority Research Program (XDB08020302) and the “135” project of the Chinese Academy of Sciences (CAS). Z.L. received the “National Thousand Young Talents” award from the Office of Global Experts Recruitment in China.

Author information

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Authors

Contributions

H.Y. carried out cloning, protein expression and purification, crystallization, X-ray data collection, structure determination and analysis, lipid blot assay, chemical cross-linking and K+ flux activity assay on C. elegans TRIC-B1; M.H. conducted molecular biology, biochemistry and crystallographic works on C. elegans TRIC-B2, performed tryptophan fluorescence spectroscopy and disulfide cross-linking experiments on C. elegans TRIC-B1, and participated in electrophysiological experiments. J.G. performed electrophysiological data recording and carried out cysteine accessibility assays. M.H. and X.O. prepared SUV and GUV samples. X. O. examined and analysed the electrophysiological data. T.C. carried out mass spectrometry experiments on extracted lipid samples. Z.L. conceived and coordinated the project, and participated in structure refinement and analysis. The manuscript was written by H.Y., M.H. and Z.L.

Corresponding author

Correspondence to Zhenfeng Liu.

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The authors declare no competing financial interests.

Additional information

Reviewer Information Nature thanks M. Prakriya and F. Van Petegem for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 Function, regulation and phylogenesis of TRIC channels.

a, A schematic drawing on the function and regulation of TRIC channels during Ca2+ release from SR/ER. The efflux of Ca2+ from ER/SR lumen into cytosol through the ryanodine receptor (RyR) or inositol triphosphate receptor (IP3R) may have dual regulatory effects on the function of TRIC channels. The micro-domain of high Ca2+ concentration near the cytosolic mouth of RyR/IP3R potentially has a stimulatory effect on TRIC channel activity, while the increase of SR/ER membrane potential (Δψ) may serve to activate TRIC channels. When activated, the TRIC channels provide K+ influx current to counteract the negative potential (within SR/ER lumen) arising from Ca2+ efflux through RyR or IP3R, and thus facilitate the Ca2+ release processes by restoring the balance of membrane potential. b, Phylogenetic analysis of the various TRIC channels from different species. The sequences of different TRIC homologues from various animal species across the taxa were retrieved from the NCBI GenBank. The phylogeny of these homologues were analysed by using DNASTAR Lasergene and Clustal W programs, and the phylogenetic tree was illustrated by using Evolview online. TRIC-B1 and -B2 are highlighted in red and both belong to the TRIC-B subfamily. c, Alignment of the TRIC-B1/2 sequences with those of Mouse TRIC-A and -B (MmTRIC-A and MmTRIC-B). The alignment result was obtained by using the Clustal W and DNAMAN programs.

Extended Data Figure 2 Identification PIP2 in C. elegans TRIC-B1/B2 and characterization of its binding site.

a, The well-defined sigmaA-weighted 2FoFc electron density of PIP2 molecule in TRIC-B2 at 2.3 Å resolution. The map is contoured at 1.2σ. b, 2FoFc simulated annealing omit map of TRIC-B2 showing the densities around PIP2 molecule. To avoid model bias, the PIP2 model was omitted in the structure used for map calculation. The contour level is 1.0σ. c, Mass spectroscopy analysis of the most abundant 34:1-PIP2 species extracted from TRIC-B2 sample. The PIP2 molecules were methylated before being separated in mass spectroscopy. Besides 34:1-PIP2, others like 34:2, 34:0, 36:2 and 36:1-PIP2 are also detected. d, The anomalous difference Fourier peaks of the phosphorus atoms on the head groups of PIP2 moleclue in TRIC-B1. The anomalous data was collected at 3 Å wavelength and the map at 4.0 Å resolution is contoured at 3.0σ. e, Lipid blot assay on the wild-type and K129A/R133L TRIC-B1 proteins. Left, a test on the reactivity of PIP2-specific antibody towards various pure phospholipid samples. PIP2, phosphatidylinositol-4,5-biphosphate; PI, phosphatidylinositol; CL, cardiolipin; PE, phosphatidylethanolamine; PS, phosphatidylserine; PA, phosphatidic acid; PG, phosphatidylglycerol; LPC, lysophosphatidylcholine. Note that only PIP2 on the blot is recognized by this highly specific antibody. Right, using the PIP2-specific antibody to probe PIP2 content in the lipid extracts from the protein samples of wild-type and K129A/R133L mutant TRIC-B1. Pure PIP2 and PI are loaded as positive and negative controls. The 1 × loading corresponds to the amount of PIP2 lipid extracted from about 7.5 μg protein. f, The PIP2-binding site within the TRIC-B1 channel is buried inside each monomer. Its head group is directly located on the surface of the cytoplasmic vestibule of the pore. g, The PIP2 in the Kir2.2 channel binds to the peripheral region near its cytoplasmic gate (Protein Data Bank code: 3SPI). The blue arrows in f and g indicate the estimated pore regions. h, i, Cartoon diagrams showing the relative positions of PIP2-binding sites on TRIC (h) and Kir (i) channels. The red stars highlight the locations of PIP2-binding residues on a pore-lining helix of the TRIC channel, or on the peripheral regions adjacent to the pore-lining helix of Kir2.2 channel.

Extended Data Figure 3 Locations of cation binding sites on C. elegans TRIC-B1 trimer.

a, b, The binding sites of Cs+ ions on TRIC-B1. The red meshes are the FCsFNa isomorphous difference Fourier peaks showing the major (Cs1) and minor (Cs2 and Cs3) binding sites of Cs+. Cs2′ indicates the peak at the Cs2 site in the symmetry-related monomer. The map is contoured at 3.7σ. The data were collected at 1.9 Å wavelength and the map resolution is 4 Å. The blue mesh shows the anomalous peak of Cs+ contoured at 3σ, matching well with the major site Cs1 in the isomorphous difference map. The anomalous peaks on the other two minor sites are below 3σ, presumably owing to the low occupancy (or low affinity) of Cs+ on these sites. c, The binding sites of Rb+ on 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 (be).

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). bd, 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, 2FoFc 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 NH group of Trp180. The 2FoFc map in blue is contoured at 1.5σ. c, d, Tryptophan fluorescence emission spectra of wild-type and W180A TRIC-B1 measured under various [Ca2+]. The error bars indicate the standard errors of the mean values (n = 10). The molar concentration of W180A mutant protein (6.9 μM) is identical to the wild-type protein sample used for the measurement. The excitation wavelength is at 295 nm. e, The difference spectra of the wild type minus W180A. The contribution of two other Trp residues (Trp62 and Trp101) is minimized in these difference spectra, and most of the signals are thereby attributed to Trp180. f, Peak 1/peak 2 intensity ratio (black) and integrated fluorescence intensity (red, 305–345 nm range, normalized) plotted against [Ca2+]. The data are extracted from the difference spectra shown in e. g, The two different conformations of M5–6 loop in respect to a nearby residue on an amphipathic helix between M1 and M2. The Asn185–Ala49′ pair in TRIC-B1 (green) and the corresponding His186–Asp50′ pair in TRIC-B2 (magenta) are highlighted as sphere models. The double-headed arrow indicates the predicted conformational switch between two states. h, Cross-linking of A49C/N185C mutant of TRIC-B1 protein in the absence (10 mM EGTA) or presence of Ca2+ (10 mM CaCl2). N185C site is located on the mobile M5–6 loop, while A49C is on a nearby region relatively invariable. i, Plot of the relative fraction of trimeric bands against diamide concentration. The error bars indicate the s.e.m. values derived from three independent repeats (n = 3). *P <0.05 (Student’s t-test). j, Cross-linking of A49C/N185C/W180A mutant of TRIC-B1 protein in the absence (10 mM EGTA) or presence of Ca2+ (10 mM CaCl2). W180A mutation was introduced to monitor the role of Trp180 in binding and responding to Ca2+. k, Plot of the relative fraction of trimeric bands of A49C/N185C/W180A mutant against diamide concentration. The error bars indicate the s.e.m. derived from three independent repeats (n = 3). l, Comparing the effects of Ca2+ on the cross-linking efficiencies of A49C/N185C and A49C/N185C/W180A mutants. Δ [I(trimer)/I(total)] indicates the difference of I(trimer)/I(total) ratio between the samples without Ca2+ (with 10 mM EGTA) and with 10 mM Ca2+. I(trimer) indicates the integrated intensities of trimeric band, while I(total) is the summed intensities of monomeric, dimeric and trimeric bands.

Extended Data Figure 9 The putative voltage-sensing helix in C. elegans TRIC-B1 compared to those in the canonical voltage-sensing domains (VSDs).

a, The charge property of the M4 helix as the potential voltage-sensing helix in TRIC-B1. b, The S4 helix of the VSD in the Kv1.2-Kv2.1 paddle chimaera channel structure. PDB code: 2R9R. The pink region is the pore domain that is covalently linked to the VSD. c, The S4 helix of the VSD in the Hv1 chimaeric channel structure. PDB code: 3WKV. d, The S4 helix of the VSD in the Ciona intestinalis voltage-sensitive phosphatase. PDB code: 4G7V. For all four structures, the voltage-sensing helices and the conserved basic amino acid residues on it are coloured cyan and blue, while those acidic groups involved in stabilizing them are in yellow and red. The other parts of the VSD are in silver.

Extended Data Table 1 Statistics of the X-ray crystallographic data of C. elegans TRIC-B1/B2

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Yang, H., Hu, M., Guo, J. et al. Pore architecture of TRIC channels and insights into their gating mechanism. Nature 538, 537–541 (2016). https://doi.org/10.1038/nature19767

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