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 Ca 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.

TRPC channels: Structure, function, regulation and recent advances in small molecular probes

“…Therefore, structure determination of membrane proteins, particularly various ion channels, is greatly facilitated [see recent review by . The current cryo-EM structures include homomeric TRPC3, TRPC4, TRPC5 and TRPC6 (Duan et al, 2019;Fan et al, 2018;Vinayagam et al, 2018;Duan et al, 2018;Tang et al, 2018;Azumaya, Sierra-Valdez, Cordero-Morales, & Nakagawa, 2018;Sierra-Valdez, Azumaya, Romero, Nakagawa, & Cordero-Morales, 2018). The overall architectures of these TRPCs are similar, all showing tetrameric structures with six transmembrane α helices in each subunit and the cytoplasmic N-termini surrounding the C-termini.…”

“…This region is halfway embedded in the membrane, with its N-terminal end exposed to the cytoplasmic side to connect with a long stretch of tightly folded linker helices located at the proximal N-terminus of each protomer. Immediately before the linker helices are the four ankyrin-like repeats that form the outskirt of the cytoplasmic architecture, which completely surrounds the four helical bundle composed of the second C-terminal helix (CH2) as designated by some (Azumaya et al, 2018;Tang et al, 2018), which is also referred to coiled-coil (Duan et al, 2018;Duan et al, 2019) or pore helix (Fan et al, 2018) domains by other groups, from the four protomers running in parallel. The CH2 domain is preceded by CH1 (also known as connecting helix (Duan et al, 2018, Duan et al, 2019 or rib helix (Vinayagam et al, 2018)) via a short loop that crosses over the adjacent protomer such that the CH2 domain is in close contact with the ankyrin repeats of the same protomer.…”

“…Open structures and structures with CIRB motif binding partners will likely reveal important insights into how relative motions between CH1, TRP domain, and S4-S5 linker, which likely interacts with the TRP domain, are involved in TRPC channel gating. Notably, at the exposed side connected to the unresolved region, the CH1 domain begins ~3-4 residues (one turn) earlier in TRPC4/5 than in TRPC3/6 [compare Vinayagam et al, 2018;Duan et al, 2019;with Tang et al, 2018;Azumaya et al, 2018]. This may give rise to the wider overall shape of TRPC4/5 (100 to 105 Å in diameter) (Duan et al, 2018;Vinayagam et al, 2018) and the skinner look of TRPC3/6 (75-85 Å in diameter) (Fan et al, 2018;Tang et al, 2018) (Fig.…”

Post-Translational Modification and Natural Mutation of TRPC Channels

“…Recently, the protein structure of TRPC3/4/5/6 have been resolved by cryo-EM with an atomic resolution [81,140,[169][170][171]. The detailed 3D structure offers a new way to evaluate potential residues that are critical for the gating and/or permeation of TRPCs.…”

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.…”