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• W1981103237 abstract "Stim and Orai proteins comprise the molecular machinery of Ca2+ release-activated Ca2+ (CRAC) channels. In T-lymphocytes, Orai1 is the pore-forming subunit and STIM1 serves as the ER Ca2+-sensor that opens Orai1 following ER Ca2+-store depletion. We are investigating mutations within Orai1 TM1 that alter channel activity in transfected HEK cells. At the extracellular side of TM1, we and others had previously shown that E106 confers Ca2+ selectivity on the STIM1-operated Orai1 current. We now show that R91, the site of the R91W mutation that causes severe combined immune deficiency, forms a very narrow part of the conducting pore at the cytosolic side of TM1. Orai1 R91C when co-expressed with STIM1 was activated normally by Ca2+ store depletion. However, treatment with diamide, a thiol-oxidizing agent, induced formation of disulfide bonds between R91C residues in adjacent Orai1 subunits. Moreover, diamide rapidly blocked STIM1-operated Ca2+ current, and current recovered during treatment with a reducing agent. In the middle of TM1, mutation of a conserved glycine residue to alanine (G98A) prevented STIM1-induced channel activity. Interestingly, mutation to aspartate (G98D) caused constitutive channel activation in a STIM1-independent manner to form a non-selective Ca2+-permeable conductance that was relatively resistant to block by Gd3+ (310 nM Kd vs 7 nM in wild-type Orai1). Moreover, the double mutant R91WG98D was also constitutively active, overcoming the normal inhibition of channel activity by tryptophan at the 91 position; and the double mutant R91CG98D was resistant to diamide block. We tentatively propose that R91 forms the gate of the Orai1 channel at the narrow inner mouth of the channel, G98 functions as a gating-hinge, and E106 promotes selective conduction of Ca2+. The channel pore is widened and ion selectivity perturbed by a negative charge at the G98 site." @default.
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• W1981103237 date "2011-02-01" @default.
• W1981103237 modified "2023-09-30" @default.
• W1981103237 title "Activation of Orai1 Channels by Mutation of a Conserved Glycine Residue in TM1" @default.
• W1981103237 doi "https://doi.org/10.1016/j.bpj.2010.12.1213" @default.
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