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

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Ca 2+ release-activated Ca 2+ (CRAC) channels are activated by direct physical interactions between Orai1, the channel protein, and STIM1, the endoplasmic reticulum Ca 2+ sensor. A hallmark of CRAC channels is fast Ca 2+ -dependent inactivation (CDI) which provides negative feedback to limit Ca 2+ 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 Ca 2+ -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 Ca 2+ 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.

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  1. eLife assessment

    This manuscript reports novel and important findings on the mechanisms of regulation of CRAC channels. Collectively, the work represents an important conceptual advancement, showing that stromal interaction molecule-1 is not necessary for Ca2+-dependent inactivation of the Orai1 channel and that Orai1 likely contains a Ca2+ sensor for autoregulatio. The experiments are carefully conducted, and the data is of high quality and support the major conclusions of the authors.

  2. Reviewer #1 (Public Review):

    In this report, Yeung et al studied a mutation in Orai1 channels (L138F) that is associated with constitutive CRAC channel activity and tubular aggregate myopathy (TAM) in humans. They put forth a model whereby substitution with large amino acids at position L138 on TM2 or the neighboring T92 on TM1 causes a steric clash between TM1 and TM2 and elicits a highly Ca2+ selective current in the absence of STIM1, the ER Ca2+ sensor protein that is the physiological activator of Orai channels. The authors went on to study one typical biophysical property of Orai1-mediated CRAC channels which is the fast Ca2+-dependent inactivation (CDI), after the surprising finding of the presence of CDI in CRAC currents mediated by T92 and L138 Orai1 mutants in the absence of STIM1. The authors showed differences in CDI between WT and mutants when using weak vs strong buffers and through computation and experimentation, they show that the Orai1 mutants have enhanced cytosolic Ca2+ sensitivity, which could be normalized when STIM1 was present. The experiments are carefully conducted and the manuscript is clearly written. The study has significant novelty and impact.

  3. Reviewer #2 (Public Review):

    The manuscript "A human tubular aggregate myopathy mutation unmasks STIM1-independent rapid inactivation of Orai1 channels" describes the effects of a disease-related gating checkpoint at the TM1-TM2 interface. The authors suggest that the mutation of one of the two oppositely located positions T92 - L138 into a large amino acid leads to constitutive activity due to steric clash. Notably, the mutants also exhibit robust Ca2+ dependent inactivation (CDI) suggesting that this feature is intrinsic to the Orai1 channel, and not as previously thought a key process that is triggered by STIM1. Nevertheless, STIM1 is able to fine-tune Ca2+ selectivity and CDI.

    This study provides an extensive electrophysiological characterization of the tubular aggregate myopathy (TAM)-disease-related Orai1 L138F mutation and based on mutational studies provides compelling evidence that constitutive activity is caused by a steric clash between TM1/TM2 Orai helices. Additionally, yet unexpectedly, the constitutive Orai1 mutants exhibit CDI behavior which is thoroughly characterized by experiments using various intracellular Ca2+-buffering reagents. By this, it is proposed that the Orai1 T92W mutant shows increased sensitivity to intracellular Ca2+. This is further revealed in a sophisticated tow step protocol, which would profit from additional control experiments. The unusual behavior of the T92W Orai1 mutant is "corrected" to that of the Orai1 wild-type form by the presence of STIM1.

  4. Reviewer #3 (Public Review):

    In this paper, Yeung et al., use patch-clamp electrophysiology measurements combined with structural analyses and mutagenesis to compellingly reveal how the tubular aggregate myopathy (TAM)-associated Orai1 L138F mutation leads to the gain of CRAC channel function. They discover that L138F not only constitutively activates Orai1-composed channels but also enhances Ca2+-dependent inactivation (CDI). The authors find that the L138F gain of function occurs due to a steric clash with T92 from an adjacent subunit and that introduction of a bulky residue at the T92 position similarly activates CRAC channels and enhances CDI in the absence of STIM1. Nevertheless, co-expression of STIM1 with strongly activating T92W or L138F mutants regularized the CDI to wild-type levels. Collectively, the work represents an important conceptual advancement, exposing that STIM1 is not necessary for CDI and that Orai1 likely contains the Ca2+ sensor intrinsically for this phenomenon.

    The authors use rigorous and careful electrophysiological measurements to probe how the TAM-related mutation (L138F) affects the biophysical properties of CRAC channels. The extensive and systematic mutagenesis (i.e. substitution to every possible amino acid at the T92 and L138 sites) coupled with these functional assessments reveal a steric clash between L138F and T92 and provide a complete picture of how any residue type at the so-called T92/L138 lever point may contribute to constitutive CRAC and CDI activity. The use of available high-resolution structural data to interpret functional data, rationalize the consequence of new mutations related to the mechanisms of L138F dysfunction, and generate new hypotheses is a strength of the research. Overall, the work provides a considerable conceptual advance in terms of understanding the molecular requirements for CRAC and CDI activity; in particular, the discovery that CDI can occur independently of STIM1 and the notion that Orai1 may contain an intrinsic Ca2+ sensor that regulates CDI are important steps forward for the field.

    While the work provides a phenomenological advancement regarding CRAC channel regulation and pinpoints new important residues for function, some aspects of the study appear incomplete. It was shown that STIM1 can normalize the enhanced CDI caused by the T92W mutation, but it is not clear how this happens. Further, the authors propose a "push" - "pull" mechanism for the complementary roles L138 and H134 in channel regulation but do not provide any structural dynamics data to support this idea. The authors provide a mathematical explanation for chelator-specific differences in CDI observed for the T92W compared to WT Orai1 but do not show any fitted data to accompany and support the model. Finally, the authors show that a considerable portion of the CDI can be eliminated after a C-terminal Orai1 deletion (i.e. residues 267-301) and probe the idea that N-terminal W76, Y80, and R83 residues may contribute to the residual CDI effect; however, after W76E, Y80E, R83E mutations showed enhanced CDI (rather than suppressed) in the context of the T92W mutation, no further experiments were pursued to account for the residual CDI.

    Overall, the strengths far outweigh the weaknesses of this study, and the conclusions drawn based on the data are compelling. The work represents an important conceptual advancement as future studies can now steer towards identifying the STIM-independent Ca2+ sensor underlying the CDI of CRAC channels and revealing structural mechanisms by which Ca2+ sensing leads to pore closure.