TRPC3 governs the spatiotemporal organization of cellular Ca2+ signatures by functional coupling to IP3 receptors

Abstract

Communication between TRPC channels and IP₃ receptors (IP₃R) is considered pivotal in the generation of spatiotemporal Ca²⁺signaling patterns. Here we revisited the role of TRPC3-IP₃R coupling for local Ca²⁺ signaling within TRPC3-harbouring micro/nanodomains using R-GECO as a reporter, fused to the channel´s C-terminus. Cytoplasmic Ca²⁺ changes at TRPC3 originated from both the entry of Ca²⁺ through the TRPC channel and Ca²⁺ mobilization via IP₃R. Local Ca²⁺ changes at TRPC3 channels expressed in HEK293 cells were predominantly biphasic with IP₃R-dependent initial Ca²⁺ transients, while exclusively monophasic signals were recorded when all three IP₃R isoforms were lacking. Abrogation of Ca²⁺ entry through TRPC3 by point mutations, which impair Ca²⁺ permeability (E630Q), cation permeation (E630K), or DAG sensitivity (G652A), promoted microdomain Ca²⁺ oscillations. Ca²⁺ signals at E630Q, E630K, and G652A channels featured initial Ca²⁺ transients along with oscillatory activity. Similarly, when extracellular Ca²⁺ was omitted, IP₃R-mediated Ca²⁺ transients and Ca²⁺ oscillations were promoted at the cytoplasmic face of wild-type TRPC3 channels. By contrast, oscillations, as well as initial Ca²⁺ transients, were virtually lacking, when the TRPC3 channels were sensitized by preexposure to low-level PLC activity. TIRF imaging provided evidence for dynamic colocalization of TRPC3 and IP₃R. We suggest that TRPC3-mediated Ca²⁺ entry controls IP₃R activity at ER-PM junctions to determine Ca²⁺ signaling signatures and enable specificity of downstream signaling.

Keywords: ER-PM nanojunction; IP3R; Phospholipase C signaling; TRPC3.