Dissecting gating mechanisms of Orai calcium channel paralogs using constitutively active Orai mutants that mimic STIM1-gated state

In humans, there are three paralogs of the Orai Ca2+ channel, which lie at the heart of the store-operated calcium entry (SOCE) machinery. While the STIM-mediated gating mechanism of Orai channels is still being actively investigated, several artificial and natural variants are known to cause constitutive activity of the human Orai1 channel. Surprisingly, little is known about the conservation of the gating mechanism among the different human Orai paralogs and orthologs in other species. In our work, we show that the mutation corresponding to the activating mutation H134A in transmembrane helix 2 (TM2) of human Orai1 also activates Orai2 and Orai3, likely via a similar mechanism. However, this cross-paralog conservation does not apply to the "ANSGA" nexus mutations in TM4 of human Orai1 which mimic the STIM1-activated state of the channel. Investigating the mechanistic background of these differences, we identified two positions, H171 and F246 in human Orai1, which directly control the channel activation triggered by the "ANSGA" mutations in Orai1. Our results shed new light on these important gating checkpoints and show that the gating mechanism of the Orai channels is affected by multiple factors that are not necessarily evolutionarily conserved, such as the TM4-TM3 coupling.