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 Ca2+ release-activated Ca2+ (CRAC) channel Orai1, Orai1 alpha and Orai1 beta, in mammalian cells arises the question whether they exhibit different functional characteristics. Orai1 alpha and Orai1 beta differ in the N-terminal 63 amino acids, exclusive of Orai1 alpha, and show different sensitivities to Ca2+-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 Orai1 alpha and Orai1 beta are required for the maintenance of regenerative Ca2+ oscillations, while TRPC1 plays a role in agonist-induced Ca2+ influx but is not essential for Ca2+ oscillations. Using APEX2 proximity labeling, co-immunoprecipitation and the fluorescence of G-GECO1.2 fused to Orai1 alpha our results indicate that agonist stimulation and Ca2+ store depletion enhance Orai1 alpha-TRPC1 interaction. Orai1 alpha is essential for TRPC1 plasma membrane location and activation. Thus, TRPC1 function in HeLa cells depends on Ca2+ influx through Orai1 alpha exclusively.

Automated summary

The two variants of the Orai1 subunit, Orai1 alpha and Orai1 beta, exhibit different functional characteristics and interactions in mammalian cells. Orai1 alpha is essential for the activation and membrane location of TRPC1, while Orai1 beta plays a role in maintaining Ca2+ oscillations.