ORAI1 channel gating and selectivity is differentially altered by natural mutations in the first or third transmembrane domain. (28th November 2018) - Bulla, M.
Abstract : Key points: Gain‐of‐function mutations in the highly selective Ca 2+ channel ORAI1 cause tubular aggregate myopathy (TAM) characterized by muscular pain, weakness and cramping. TAM‐associated mutations in ORAI1 first and third transmembrane domain facilitate channel opening by STIM1, causing constitutive Ca 2+ influx and increasing the currents evoked by Ca 2+ store depletion. Mutation V107M additionally decreases the channel selectivity for Ca 2+ ions and its inhibition by acidic pH, while mutation T184M does not alter the channel sensitivity to pH or to reactive oxygen species. The ORAI blocker GSK‐7975A prevents the constitutive activity of TAM‐associated channels and might be used in therapy for patients suffering from TAM. Abstract: Skeletal muscle differentiation relies on store‐operated Ca 2+ entry (SOCE) mediated by STIM proteins linking the depletion of endoplasmic/sarcoplasmic reticulum Ca 2+ stores to the activation of membrane Ca 2+ ‐permeable ORAI channels. Gain‐of‐function mutations in STIM1 or ORAI1 isoforms cause tubular aggregate myopathy (TAM), a skeletal muscle disorder with muscular pain, weakness and cramping. Here, we characterize two overactive ORAI1 mutants from patients with TAM: V107M and T184M, located in the first and third transmembrane domain of the channel. When ectopically expressed in HEK‐293T cells or human primary myoblasts, the mutated channels increased basal and store‐operated Ca 2+ entry. The constitutive activity of V107M, L138F, T184M and P245L mutants was prevented by low concentrations of GSK‐7975A while the G98S mutant was resistant to inhibition. Electrophysiological recordings confirmed ORAI1‐V107M constitutive activity and revealed larger STIM1‐gated V107M‐ and T184M‐mediated currents with conserved fast and slow Ca 2+ ‐dependent inactivation. Mutation V107M altered the channel selectivity for Ca 2+ ions and conferred resistance to acidic inhibition. Ca 2+ imaging and molecular dynamics simulations showed a preserved sensitivity of T184M to the negative regulation by reactive oxygen species. Both mutants were able to mediate SOCE in Stim1 −/− /Stim2 −/− mouse embryonic fibroblasts expressing the binding‐deficient STIM1‐F394H mutant, indicating a higher sensitivity for STIM1‐mediated gating, with ORAI1‐T184M gain‐of‐function being strictly dependent on STIM1. These findings provide new insights into the permeation and regulatory properties of ORAI1 mutants that might translate into therapies against diseases with gain‐of‐function mutations in ORAI1 . Key points: Gain‐of‐function mutations in the highly selective Ca 2+ channel ORAI1 cause tubular aggregate myopathy (TAM) characterized by muscular pain, weakness and cramping. TAM‐associated mutations in ORAI1 first and third transmembrane domain facilitate channel opening by STIM1, causing constitutive Ca 2+ influx and increasing the currents evoked by Ca 2+ store depletion. Mutation V107M additionally decreases the channel selectivity for Ca 2+ ions and its inhibition by acidic pH, while mutation T184M does not alter the channel sensitivity to pH or to reactive oxygen species. The ORAI blocker GSK‐7975A prevents the constitutive activity of TAM‐associated channels and might be used in therapy for patients suffering from TAM.