TRPC3 governs the spatiotemporal organization of cellular Ca signatures by functional coupling to IP receptors.

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