A human tubular aggregate myopathy mutation unmasks STIM1-independent rapid inactivation of Orai1 channels

Abstract

Ca2+ release-activated Ca2+ (CRAC) channels are activated by direct physical interactions between Orai1, the channel protein, and STIM1, the endoplasmic reticulum Ca2+ sensor. A hallmark of CRAC channels is fast Ca2+-dependent inactivation (CDI) which provides negative feedback to limit Ca2+ entry through CRAC channels. Although STIM1 is thought to be essential for mediating CDI, the molecular mechanism of CDI remains largely unknown. Here, we examined a gain-of-function (GOF) human Orai1 disease mutation, L138F, that causes tubular aggregate myopathy (TAM). Through pairwise mutational analysis, we determine that large amino acid substitutions at either L138 or the neighboring T92 locus evoke highly Ca2+-selective currents in the absence of STIM1. We find that the GOF phenotype arises due to steric clash between L138 on TM2 and T92 located on the pore helix. Surprisingly, strongly activating L138 and T92 mutations also show CDI in the absence of STIM1, contradicting prevailing views that STIM1 is required for inactivation. CDI of constitutively open T92W and L138F mutants occurred with similar kinetics as WT Orai1 but showed enhanced intracellular Ca2+ sensitivity, which could be normalized by the addition of STIM1. Truncation of the Orai1 C-terminus reduced T92W CDI consistent with a key role for the Orai1 C-terminus for CDI. Overall, these results elucidate the molecular basis of the human TAM-linked mutation and indicate that CDI of CRAC channels is mediated by an Orai1-intrinsic mechanism with STIM1 tuning the calcium sensitivity of CDI.