Structural and Functional Analyses of TRPC3 Reveal Allosteric Gating Modulation by the Cytoplasmic Domain

Abstract

TRPC3 is associated with neurodegenerative diseases, memory loss, and hypertension. In blood vessels, TRPC3 is an essential component of the cellular mechanisms by which vasoconstrictors regulate blood pressure. Vasoconstrictors, such as angiotensin II, bind to phospholipase C (PLC)-coupled receptors, increasing the intracellular levels of DAG, which in turn activates TRPC3. Upon activation, TRPC3 channels induce depolarization increases intracellular Ca2+and promotes vasoconstriction. Here, we show the full-length and cytoplasmic domain (CPD) cryoEM structures for human TRPC3 in the apo state at 5.8 and 4.0 Å resolution. A TRPC3 subunit consists of an N-terminus formed by four ankyrin repeats, the linker helical domain (LHD1-9), and the pre-S1 elbow. The N-terminus is followed by six membrane-spanning helices (S1-S6) and a pore helix connecting to a re-entrant loop that forms the selectivity filter. The TRP helix is immediately adjacent to S6, and the C-terminus consists of two alpha-helices connected by a small linker. TRPC3 transmembrane domain resembles those of other TRP channels, whereas the CPD features major differences. A C-terminal domain swap occurs at the center of the CPD, where horizontal helices (HH) transition into a coiled-coil bundle, highlighting a unique fold for the TRPC subfamily. Comparison of TRPC3 structures reveals that the HH can reside in two distinct positions. Electrophysiological analyses demonstrate that TRPC3 activity increases by shortening the length of the C-terminal loop connecting the HH with the TRP helices; likewise elongating the length of the loop has the opposite effect. Our findings demonstrate that the C-terminal loop impacts channel gating by altering the allosteric coupling between the cytoplasmic and transmembrane domains. Future functional and structural experiments are underway to validate whether the upward movement of the HHs corresponds to a positive modulation of the TRPC3 gating cycle.