Graphical AbstractOrai1 and Orai3 channel activation depends in an isoform-specific manner on two non-conserved residues in TM3 (Orai1: V181, L185, Orai3: A156, F160). Mutation of these residues to alanine leads in the absence of STIM1 to small constitutive activity of the respective Orai1 mutants, however, to huge constitutive currents of the respective Orai3 mutants. Overall, two non-conserved residues in TM3 control the maintenance of the closed state as well as an opening permissive conformation of Orai channels in an isoform-specific manner.