Orai1α, but not Orai1β, co-localizes with TRPC1 and is required for its plasma membrane location and activation in HeLa cells

The identification of two variants of the canonical pore-forming subunit of the Ca(2+) release-activated Ca(2+) (CRAC) channel Orai1, Orai1α and Orai1β, in mammalian cells arises the question whether they exhibit different functional characteristics. Orai1α and Orai1β differ in the N-terminal 63 amino acids, exclusive of Orai1α, and show different sensitivities to Ca(2+)-dependent inactivation, as well as distinct ability to form arachidonate-regulated channels. We have evaluated the role of both Orai1 variants in the activation of TRPC1 in HeLa cells. We found that Orai1α and Orai1β are required for the maintenance of regenerative Ca(2+) oscillations, while TRPC1 plays a role in agonist-induced Ca(2+) influx but is not essential for Ca(2+) oscillations. Using APEX2 proximity labeling, co-immunoprecipitation and the fluorescence of G-GECO1.2 fused to Orai1α our results indicate that agonist stimulation and Ca(2+) store depletion enhance Orai1α–TRPC1 interaction. Orai1α is essential for TRPC1 plasma membrane location and activation. Thus, TRPC1 function in HeLa cells depends on Ca(2+) influx through Orai1α exclusively. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00018-021-04098-w.