Cryo-EM structure of the cytoplasmic domain of murine transient receptor potential cation channel subfamily C member 6 (TRPC6)

Abstract

The kidney maintains the internal milieu by regulating the retention and excretion of proteins, ions, and small molecules. The glomerular podocyte forms the slit diaphragm of the ultrafiltration filter, whose damage leads to progressive kidney failure and focal segmental glomerulosclerosis (FSGS). The canonical transient receptor potential 6 (TRPC6) ion channel is expressed in the podocyte, and mutations in its cytoplasmic domain cause FSGS in humans. In vitro evaluation of disease-causing mutations in TRPC6 has revealed that these genetic alterations result in abnormal ion channel gating. However, the mechanism whereby the cytoplasmic domain modulates TRPC6 function is largely unknown. Here, we report a cryo-EM structure of the cytoplasmic domain of murine TRPC6 at 3.8 Å resolution. The cytoplasmic fold of TRPC6 is characterized by an inverted dome-like chamber pierced by four radial horizontal helices that converge into a vertical coiled-coil at the central axis. Unlike other TRP channels, TRPC6 displays a unique domain swap that occurs at the junction of the horizontal helices and coiled-coil. Multiple FSGS mutations converge at the buried interface between the vertical coiled-coil and the ankyrin repeats, which form the dome, suggesting these regions are critical for allosteric gating modulation. This functionally critical interface is a potential target for drug design. Importantly, dysfunction in other family members leads to learning deficits (TRPC1/4/5) and ataxia (TRPC3). Our data provide a structural framework for the mechanistic investigation of the TRPC family.

Keywords: TRPC6; calcium channel; cryo-electron microscopy; electrophysiology; glomerulosclerosis; ion channel; kidney; membrane protein; purification; transient receptor potential channels (TRP channels); vasoconstriction.

Figure 1.

Functional and biochemical characterization of mTRPC6. A and B, OAG-evoked currents of HEK293 cell-expressing Δ94TRPC6 (referred to as TRPC6 from here) and WT TRPC6, as determined by whole-cell patch-clamp recording. C, size-exclusion chromatography profile of PMAL-C8 TRPC6 protein. Inset, stained protein on the SDS-PAGE gel corresponds to the size of the purified channel monomers.

Figure 2.

Projection structures of TRPC6. A, representative negative stain 2D class averages showing the flexible and well-defined domains of TRPC6. B, a representative raw micrograph of TRPC6 in vitrified ice recorded using a Titan Krios microscope. C, representative 2D class averages of vitrified TRPC6 particles. The number of particles contained in each class is indicated in the bottom right corner.

Figure 3.

Cryo-EM structure and atomic model of TRPC6. A, secondary structure organization of TRPC6. B, the structure of a single subunit. The substructures are color-coded in the same way as in A. C, EM density map showing the tetrameric organization of the cytoplasmic domain. Each subunit is represented by different colors. The overall resolution is 3.8 Å based on Fourier shell correlation = 0.143, seen in E. D, ribbon diagram of the atomic model generated from the EM density map. E, Fourier shell correlation (FSC) curve for the electron density map calculated from the Titan Krios data collected for TRPC6.

Figure 4.

Unique arrangements of the C-terminal α-helices in TRPC6. A and B, top view of EM density map and cylinder representations of TRPC6 C-terminal segment domain swap. B, arrows indicate the direction of the swap. C, EM density map of the domain swap visualized at two different map thresholds. The linker is clearly resolved in our map. Yellow sphere represents the unassigned density observed when the reconstruction was calculated from the Titan Krios dataset. D, comparison of TRP channels HH and VH helices arranged in a coiled-coil structure, PDB: 6BCL, 6BPQ, and 3J9P. Note that TRPA1 lacks the HH.

Figure 5.

Electrostatic potential of TRPC6 cytoplasmic domain. A, side view of the cytoplasmic domain indicating an overall negative charge at the bottom of the domain and patches of positive charge at the intersections of the HH and the LHD. B, top view indicating the overall negative charge of the inner chamber. The openings are difficult to see from this view, but are present (yellow circle). C, cut-through view of the cytoplasmic domain showing an overall negative charge of the internal canal crossing from the cytoplasm to the inner chamber. D, bottom view showing the openings between the inner chamber and the cytoplasm. Blue indicates positive and partial-positive regions; red indicates negative and partial-negative regions.

Figure 6.

Interaction between the loops and horizontal helices. A and B, superposition of the EM density map and the ribbon diagram of the atomic model, highlighting the interaction between loop 1 (connecting AR1 and 2), HH, and AR4. C and D, similar representations as above, showing the interface between loop 3 (connecting AR3 and 4) and HH. In B and D, the arrow indicates the merging point in the density map that corresponds to a residue contact.

Figure 7.

Location of FSGS mutations in the TRPC6 cytoplasmic domain. A, one subunit of TRPC6 with mutations that have been identified in patients with FSGS mapped onto the 3D structure. Three different groups are labeled in red, green, and magenta (see text for details). B, group 1 mutations are labeled in red and clustered around the intersection of the ankyrin repeats, mostly AR1, and the vertical helix. Asn-109 and Tyr-895 form a strong contact in this area and are highlighted in dark gray. C, group 2 mutants are shown in green and clustered around the contact point of ankyrin repeats and the horizontal helix. Contacts between loop 1 and the HH and AR4 of the adjacent subunit, Gly-132 and Lys-863 and Tyr-130 and Tyr-231, respectively, are highlighted in dark gray.