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

Abstract

Transient receptor potential canonical (TRPC) channels constitute a group of receptor-operated calcium-permeable nonselective cation channels of the TRP superfamily. The seven mammalian TRPC members, which can be further divided into four subgroups (TRPC1, TRPC2, TRPC4/5, and TRPC3/6/7) based on their amino acid sequences and functional similarities, contribute to a broad spectrum of cellular functions and physiological roles. Studies have revealed complexity of their regulation involving several components of the phospholipase C pathway, G_i and G_o proteins, and internal Ca²⁺ stores. Recent advances in cryogenic electron microscopy have provided several high-resolution structures of TRPC channels. Growing evidence demonstrates the involvement of TRPC channels in diseases, particularly the link between genetic mutations of TRPC6 and familial focal segmental glomerulosclerosis. Because TRPCs were discovered by the molecular identity first, their pharmacology had lagged behind. This is rapidly changing in recent years owning to great efforts from both academia and industry. A number of potent tool compounds from both synthetic and natural products that selective target different subtypes of TRPC channels have been discovered, including some preclinical drug candidates. This review will cover recent advancements in the understanding of TRPC channel regulation, structure, and discovery of novel TRPC small molecular probes over the past few years, with the goal of facilitating drug discovery for the study of TRPCs and therapeutic development.

Keywords: Calcium signaling; Drug discovery; Heterotrimeric G proteins; Nonselective cation channels; Phospholipase C; Receptor-operated channels.

Fig. 1.. Structures of TRPC channels.

A & B, cryo-EM structures of C terminus truncated mouse TRPC4 (aa 1–758) (A) and full-length human TRPC6 (aa 1–931) (B), as reported by Duan et al. (2018) and Tang et al. (2018), respectively. The transmembrane regions are defined by the horizontal black lines with the thickness of about 30 Å. Areas of the Calmodulin- and IP₃ receptor-binding (CIRB) motifs and the two conserved acidic residues (EE) critical for regulation by STIM1 are indicated by the dashed red circles. The extracellular protrusion of S3 transmembrane helix in TRPC6 is encircled by brown dashed line. Note the missing structures between the TRP re-entrant loop and CIRB motif in both examples. C & D, ribbon diagrams of single subunits of TRPC4 (C) and TRPC6 (D). TRP domain (in C) is equivalent to TRP helix (in D); Connecting helix and coiled-coil domain (in C) are equivalent to C terminal helices 1 and 2 (in D), respectively. E & F, topology and domain organization of TRPC4 (E) and TRPC6 (F) single subunits. Cylinders indicate α helices; dashed lines highlight unresolved structures.

Fig. 2.. Intracellular Na⁺binding sites identified from TRPC4 (left) and TRPC5 (right) cryo-EM structures.

S2 and S3 depict the second and third transmembrane helices, respectively. Adopted from Li et al. (2019) with modifications.

Fig. 3.. Mechanism of activation of TRPC channels.

A, receptor-operated channel (ROC) activation of TRPCs. Independently of STIM1, receptor activation leads to PLC hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP₂) and production of inositol 1,4,5-trisphosphate (IP₃), diacylglycerols (DAG), and protons (H⁺). All of them have been implicated in the regulation of TRPC channel function. In addition, IP₃ activates IP₃ receptors (IP₃R) on the endoplasmic reticulum (ER) membrane to release stored Ca²⁺, causing [Ca²⁺]_c to increase. Ca²⁺ regulates TRPC channels in both calmodulin (CaM)-dependent and -independent manners. Specifically for TRPC4 and C5, the activation of G_i/o proteins also triggers channel activation in conjunction with PLC stimulation. B, store (or STIM)-operated channel (SOC) activation of TRPCs. TRPC1/4/5 have been shown to be directly activated by STIM1 (see text for details), which senses ER Ca²⁺ store depletion to oligomerize and move towards the plasma membrane (PM). This may be facilitated by junctate, which interacts with IP₃Rs, TRPCs, and STIM1, as well as by Ca²⁺ influx mediated by Orai1. C, linear representation of selected key structure features of TRPC4α (upper) and TRPC6 (lower). Ankyrin-like (ANK) repeats, transmembrane segments (blue boxes), and CaM-binding sites (red circles) are labeled. EWKFAR indicates the TRP motif. VTTRL indicates the PDZ-binding domain. Experimental evidence for some of the features indicated in TRPC4α has only been reported for TRPC5. −, experimental evidence exists for negative regulation; ±, experimental evidence exists for both positive and negative regulation. Binding motifs for NHERF (Tang et al., 2000), PIP₂ and PIP₃ (Kwon et al., 2007), SESTD1 (Miehe et al., 2010), G_i/oα-GTP (Jeon et al., 2012), STIM1 (Zeng et al., 2008), spectrin (Odell, Van Helden, & Scott, 2008), and Calmodulin- and IP₃ receptor-binding (CIRB) site (Zhang et al., 2001; Tang et al., 2001) are indicated.