Department of Physiology, University of California, Los Angeles, California 90095, USA.
J Biol Chem. 2011 Oct 7;286(40):35318-28. doi: 10.1074/jbc.M111.265884. Epub 2011 Aug 24.
Orai1, the pore subunit of Ca(2+) release-activated Ca(2+) channels, has four transmembrane segments (TMs). The first segment, TMI, lines the pore and plays an important role in channel activation and ion permeation. TMIII, on the other hand, does not line the pore but still regulates channel gating and permeation properties. To understand the role of TMIII, we have mutated and characterized several residues in this domain. Mutation of Trp-176 to Cys (W176C) and Gly-183 to Ala (G183A) had dramatic effects. Unlike wild-type channels, which exhibit little outward current and are activated by STIM1, W176C mutant channels exhibited a large outward current at positive potentials and were constitutively active in the absence of STIM1. G183A mutant channels also exhibited substantial outward currents but were active only in the presence of 2-aminoethoxydiphenyl borate (2-APB), irrespective of STIM1. With W176C mutant channels inward, monovalent currents were blocked by Ca(2+) with a high affinity similar to the wild type, but the Ca(2+)-dependent blocking of outward currents differed in the two cases. Although a 50% block of the WT outward current required 250 μm Ca(2+), more than 6 mm was necessary to have the same effect on W176C mutant channels. In the presence of extracellular Ca(2+), W176C and G183A outward currents developed slowly in a voltage-dependent manner, whereas they developed almost instantaneously in the absence of Ca(2+). These changes in permeation and gating properties mimic the changes induced by mutations of Glu-190 in TMIII and Asp-110/Asp-112 in the TMI/TMII loop. On the basis of these data, we propose that TMIII maintains negatively charged residues at or near the selectivity filter in a conformation that facilitates Ca(2+) inward currents and prevents outward currents of monovalent cations. In addition, to controlling selectivity, TMIII may also stabilize channel gating in a closed state in the absence of STIM1 in a Trp-176-dependent manner.
Orai1 是钙释放激活钙通道的孔亚基，具有四个跨膜片段 (TMs)。第一个片段 TMI 排列在孔中，对通道激活和离子渗透起着重要作用。另一方面，TMIII 不排列在孔中，但仍调节通道门控和渗透特性。为了了解 TMIII 的作用，我们对该结构域中的几个残基进行了突变和特征分析。突变色氨酸 176 为半胱氨酸（W176C）和甘氨酸 183 为丙氨酸（G183A）产生了显著的影响。与野生型通道不同，野生型通道仅在 STIM1 存在下表现出微弱的外向电流和激活，W176C 突变体通道在正电位下表现出大的外向电流并且在没有 STIM1 的情况下是组成性激活的。G183A 突变体通道也表现出相当大的外向电流，但仅在存在 2-氨基乙氧基二苯硼酸盐 (2-APB) 时才具有活性，而与 STIM1 无关。用 W176C 突变体通道进行内向，单价电流被 Ca(2+) 以类似于野生型的高亲和力阻断，但两种情况下外向电流的 Ca(2+) 依赖性阻断不同。尽管 WT 外向电流的 50%阻断需要 250 μm Ca(2+)，但对于 W176C 突变体通道，需要超过 6 mM 才能达到相同的效果。在存在细胞外 Ca(2+) 的情况下，W176C 和 G183A 外向电流以电压依赖性方式缓慢发展，而在没有 Ca(2+) 的情况下几乎瞬间发展。这些渗透和门控特性的变化类似于 TMIII 中 Glu-190 突变和 TMI/TMII 环中 Asp-110/Asp-112 突变诱导的变化。基于这些数据，我们提出 TMIII 在其选择性过滤器附近的构象中保持负电荷残基，以促进 Ca(2+) 内向电流并防止单价阳离子的外向电流。此外，除了控制选择性外，TMIII 还可能以依赖色氨酸 176 的方式稳定通道门控在 STIM1 缺失时的关闭状态。
