TRPC3 governs the spatiotemporal organization of cellular Ca2+ signatures by functional coupling to IP3 receptors
Sanja Ćurčić, Hazel Erkan‐Candag, Johannes Pilic, Roland Malli, Patrick Wiedner, Oleksandra Tiapko, Klaus Groschner
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.