Department of Physiology and Biophysics, University of California, Irvine, CA 92697, USA.
Proc Natl Acad Sci U S A. 2011 Oct 25;108(43):17838-43. doi: 10.1073/pnas.1114821108. Epub 2011 Oct 10.
Stim and Orai proteins comprise the molecular machinery of Ca(2+) release-activated Ca(2+) (CRAC) channels. As an approach toward understanding the gating of Orai1 channels, we investigated effects of selected mutations at two conserved sites in the first transmembrane segment (TM1): arginine 91 located near the cytosolic end of TM1 and glycine 98 near the middle of TM1. Orai1 R91C, when coexpressed with STIM1, was activated normally by Ca(2+)-store depletion. Treatment with diamide, a thiol-oxidizing agent, induced formation of disulfide bonds between R91C residues in adjacent Orai1 subunits and rapidly blocked STIM1-operated Ca(2+) current. Diamide-induced blocking was reversed by disulfide bond-reducing agents. These results indicate that R91 forms a very narrow part of the conducting pore at the cytosolic side. Alanine replacement at G98 prevented STIM1-induced channel activity. Interestingly, mutation to aspartate (G98D) or proline (G98P) caused constitutive channel activation in a STIM1-independent manner. Both Orai1 G98 mutants formed a nonselective Ca(2+)-permeable conductance that was relatively resistant to block by Gd(3+). The double mutant R91W/G98D was also constitutively active, overcoming the normal inhibition of channel activity by tryptophan at the 91 position found in some patients with severe combined immunodeficiency (SCID), and the double mutant R91C/G98D was resistant to diamide block. These data suggest that the channel pore is widened and ion selectivity is altered by mutations at the G98 site that may perturb α-helical structure. We propose distinct functional roles for G98 as a gating hinge and R91 as part of the physical gate at the narrow inner mouth of the channel.
刺激和 Orai 蛋白构成 Ca(2+)释放激活的 Ca(2+)（CRAC）通道的分子机制。作为理解 Orai1 通道门控的一种方法，我们研究了两个保守位点的突变对第一个跨膜片段（TM1）的影响：位于 TM1 胞质末端附近的精氨酸 91 和位于 TM1 中部附近的甘氨酸 98。当与 STIM1 共表达时，Orai1 R91C 可被 Ca(2+)储存耗竭正常激活。用二硫苏糖醇（一种硫醇氧化剂）处理可诱导相邻 Orai1 亚基中 R91C 残基之间形成二硫键，并迅速阻断 STIM1 操作的 Ca(2+)电流。二硫键还原剂可逆转二硫键诱导的阻断。这些结果表明，R91 形成了胞质侧导孔的非常狭窄的部分。甘氨酸替换 G98 可防止 STIM1 诱导的通道活性。有趣的是，突变到天冬氨酸（G98D）或脯氨酸（G98P）可导致 STIM1 非依赖性的组成型通道激活。Orai1 G98 突变体均形成非选择性 Ca(2+)渗透性电导，对 Gd(3+)的阻断相对耐受。双突变体 R91W/G98D 也呈组成型激活，克服了某些严重联合免疫缺陷（SCID）患者中第 91 位色氨酸对通道活性的正常抑制作用，双突变体 R91C/G98D 对二硫苏糖醇阻断具有抗性。这些数据表明，G98 位点的突变会加宽通道孔并改变离子选择性，可能会破坏α-螺旋结构。我们提出 G98 作为门控铰链和 R91 作为通道狭窄内口物理门的一部分具有不同的功能作用。
