Structure-function analyses of the ion channel TRPC3 reveal that its cytoplasmic domain allosterically modulates channel gating

Abstract

The transient receptor potential ion channels support Ca²⁺ permeation in many organs, including the heart, brain, and kidney. Genetic mutations in transient receptor potential cation channel subfamily C member 3 (TRPC3) are associated with neurodegenerative diseases, memory loss, and hypertension. To better understand the conformational changes that regulate TRPC3 function, we solved the cryo-EM structures for the full-length human TRPC3 and its cytoplasmic domain (CPD) in the apo state at 5.8- and 4.0-Å resolution, respectively. These structures revealed that the TRPC3 transmembrane domain resembles those of other TRP channels and that the CPD is a stable module involved in channel assembly and gating. We observed the presence of a C-terminal domain swap at the center of the CPD where horizontal helices (HHs) transition into a coiled-coil bundle. Comparison of TRPC3 structures revealed that the HHs can reside in two distinct positions. Electrophysiological analyses disclosed that shortening the length of the C-terminal loop connecting the HH with the TRP helices increases TRPC3 activity and that elongating the length of the loop has the opposite effect. Our findings indicate that the C-terminal loop affects channel gating by altering the allosteric coupling between the cytoplasmic and transmembrane domains. We propose that molecules that target the HH may represent a promising strategy for controlling TRPC3-associated neurological disorders and hypertension.

Keywords: GSK-1702934A; TRPC3; calcium channel; cryo-electron microscopy; electrophysiology; ion channel; neurotransmitter; structural biology; transient receptor potential channels (TRP channels); vascular biology.

Figure 1.

Cryo-EM structure of full-length human TRPC3 at 5.8 Å. A, size-exclusion chromatography profile of digitonin-solubilized and GDN-purified TRPC3 from Sf9 cells. Inset, stain-free protein on SDS-PAGE gel corresponds to TRPC3 monomer (97 kDa). B, left, micrograph after motion correction of TRPC3 in GDN micelles (TRPC3_GDN), taken on an FEI Polara microscope. Note that particles are monodisperse and some are circled in black. Right, representative 2D class averages of TRPC3_GDN. Particles were aligned and classified in RELION 2.1. The number of particles in each class is shown in the lower right corner of each box. C, electron density map of TRPC3_GDN tetrameric assembly. GDN micelle is denoted in light gray at a higher threshold than the four subunits, colored in blue, green, pink, and purple. D, side view of TRPC3_GDN with local resolution calculated in ResMap indicated by the heat map scale bar. High- to low-resolution runs as blue to red, from 4.0 to 8.0 Å. E, side view cross-section of the tetrameric TRPC3_GDN highlighting the hollow inner chamber below the transmembrane domain. AU, arbitrary units; ECD, extracellular domain.

Figure 2.

Cryo-EM structure of the TRPC3 CPD at 4.0 Å. A, size-exclusion chromatography profile of PMAL-C8–stabilized TRPC3 protein. Inset, stain-free protein on the SDS-PAGE gel corresponds to the size of the purified channel monomers (97 kDa). B, left, micrograph after motion correction of TRPC3 in PMAL-C8 (TRPC3_PMAL), taken on a Titan Krios. Note that particles are monodisperse and some are circled in black. Right, representative 2D class averages of TRPC3_PMAL. Particles were aligned and classified in RELION 2.1, and the number of particles in each class is shown in the lower right corner of each box. Arrows indicate the diffuse density of the TMD. C, FSC curve showing a 4.0-Å cutoff at the gold standard value of 0.143. D, electron density map of TRPC3 CPD tetrameric assembly. E, ribbon diagram of the atomic model generated from the EM density map shown in D. F, CPD ribbon diagram of a single TRPC3_PMAL subunit. The subdomains are labeled as ARD (yellow), LHD (blue), and the C-terminal HH and VH (pink). The black arrow highlights the linker between the HH and VH. AU, arbitrary units.

Figure 3.

Detailed structural features of TRPC3. A, ribbon diagram of the TRPC3 model built from the EM density map shown in Figs. 1C and 2D with the four subunits in blue, green, pink, and purple. B, cartoon representation of the secondary structure organization of a single TRPC3 subunit. ARD is in yellow, LHD is in blue, transmembrane helices are in red (S1–S4) and gray (S5, pore helix, and S6), the TRP domain helix is in green, and the C-terminal helices are in pink. Light blue represents the plasma membrane. Regions not resolved in the structure are shaded out. C, the ribbon diagram structure of a single TRPC3 subunit. The domains are color-coded in the same way as in B. D, top view of TRPC3 with HHs and VHs represented by cylinders inside the electron density map, highlighting the C-terminal helices' domain swaps. E, close-up look at the domain swap that occurs at the intersection of the HHs and VHs. The map is displayed at two threshold levels with the four-subunit map in light gray and four different subunits in colors showing the connection between the HHs and VHs. F, cylindrical representation of the intersection of the four HHs and VHs of TRPC3 (left) and TRPM4 (right; PDB code 6BCL). Note the lack of domain swap in TRPM4.

Figure 4.

Structural diversity of the CPD. Two opposite subunits of the TRPC3_GDN (A) and the TRPC3_digitonin (PDB code 6CUD) (B) structures showing S6 segments, TRP helices, HHs, and VHs. Angles between opposite TRP helices and HHs are indicated. C, close-up look comparing the angles of the HH (top) and VH (bottom) of the TRPC3_GDN and TRPC3_digitonin structures. D and E, bottom views of the TRPC3_GDN (D) and TRPC3_digitonin (E) structures. Note, that the ARDs in the TRPC3_GDN structure are closer to each other at the periphery of the CPD and to the VHs at the central axis, indicated by the double-headed arrows. F, organization of the HHs and VHs in TRPC3_GDN (top) and TRPC3_digitonin (bottom) structures. Note, that the domain swap is absent in TRPC3_digitonin. G, cartoon summarizing the differences observed in the CPDs of the TRPC3 structures.

Figure 5.

Functional characterization of TRPC3 C-terminal loop mutants. A, subunit diagram and amino acid sequence highlighting the changes made in the C-terminal loop of human TRPC3 to generate the deletion constructs Δ749–776 (Δ28TRPC3), Δ740–745 (Δ6TRPC3), and the two glycine insertion constructs TRPC3+4G and M742G+8G. Shown are representative whole-cell recordings from HEK293 cells expressing TRPC3 (B), Δ28TRPC3 (C), Δ6TRPC3 (D), TRPC3+4G (E), and M742G+8G (F). Currents were evoked by 0.2 (red) and 1 μm (blue) GSK-170 (GSK). G, box-plot summary of the ratio between the current evoked by 0.2 μm and the maximal current at 1 μm GSK-170 at +100 mV. For each construct, we measured n = 12 independent whole-cell recordings. Box plots show the mean, median, and the 75th to 25th percentiles. Statistics were calculated using a one-way analysis of variance and Bonferroni test. Error bars indicate the 1 and 99th percentiles, ** indicates p < 0.05, and *** indicates p < 0.001. Bkgrd, background.

Figure 6.

Proposed model for the role of the C-terminal loop in TRPC3 channel function. This diagram summarizes the electrophysiological data in which the C-terminal loop length modulates TRPC3 activity. A, schematic of the structure of TRPC3_GDN. A shorter loop enhances coupling between the HH and the TMD and in turn increases activity (B), whereas a longer loop has the opposite effect (C).