Discovery of novel gating checkpoints in the Orai1 calcium channel by systematic analysis of constitutively active mutants of its paralogs and orthologs

•The TM2-TM1 coupling is conserved in all human Orai channel paralogs, whereas the TM4-TM3 coupling appears to be altered.•Contrary to Orai1, introduction of the ANSGA substitution does not activate Orai2 and Orai3 channels.•The Xenopus Orai1-ANSGA mutant is constitutively active, whereas the Drosophila Orai-ANSGA is not.•Orai1 residues H171 and F246 are critical for its activation by the ANSGA substitution, but not by STIM1.•The ANSGA mutant itself may not be an accurate surrogate for the STIM1-activated Orai1 channel.