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. 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.

Mammalian TRP ion channels are insensitive to membrane stretch

“…3A,B). For these experiments we used, in addition to WT, an N-terminal truncation (Δ94) of TRPC6, whose channel properties have been reported to be very similar to the WT channel (Azumaya et al, 2018). Previously, the Δ94 TRPC6 truncation was used for the structural determination of the TRPC6 cytoplasmic domain (Azumaya et al, 2018).…”

“…Protocol for mTRPC6 purification was used from a previous publication (Azumaya et al, 2018). Two versions of mTRPC6 were purified: 94 aa truncated (Δ94 mTRPC6) and the full-length (WT) protein.…”

Structural mechanisms of transient receptor potential ion channels

“…Indeed, protonation of the luminal loop on PMD regulates TRPML channels (Li et al, 2017). In both TRPC3 and TRPC6, the S3 helix extends into the extracellular space and, together with the neighboring S1–S2 and S3–S4 loops, forms a distinct extracellular domain (Azumaya et al, 2018; Fan et al, 2018; Sierra-Valdez et al, 2018; Tang et al, 2018). This structure interacts with the channel pore, implying that it may regulate channel function upon receiving external stimuli and confer drug sensitivity.…”

Post-Translational Modification and Natural Mutation of TRPC Channels

“…G109S, N110H, Q889K, R895C, and E897K were reported to increase the basal activity of TRPC6 [137]; N143S, H218L and A404V augmented the maximum influx of Ca 2+ [137]; M132T, S270T and K874X caused delayed inactivation of TRPC6 [128,138]; R68W, P112Q enhanced the cell surface expression of the channel [127,139]. A recent study of TRPC6 protein structure from cryo-EM has provided a more direct and detailed mechanism of alterations caused by these mutations [140]. G109S, P112Q, N143S, R895C, and E897K, which are located at buried interface between the ankyrin repeats and the vertical helix, are not accessible from the outside.…”

“…They were therefore speculated to influence the internal movement of the ankyrin repeats and the coiled-coil helix instead of influencing the interaction of TRPC6 with other proteins. M132T, located at the joint interface of two adjacent subunits, was speculated to affect the intersubunit interaction [140].…”