Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Feinberg School of Medicine, Chicago, IL 60611.
J Gen Physiol. 2014 Mar;143(3):325-43. doi: 10.1085/jgp.201311108.
Prevailing models postulate that high Ca(2+) selectivity of Ca(2+) release-activated Ca(2+) (CRAC) channels arises from tight Ca(2+) binding to a high affinity site within the pore, thereby blocking monovalent ion flux. Here, we examined the contribution of high affinity Ca(2+) binding for Ca(2+) selectivity in recombinant Orai3 channels, which function as highly Ca(2+)-selective channels when gated by the endoplasmic reticulum Ca(2+) sensor STIM1 or as poorly Ca(2+)-selective channels when activated by the small molecule 2-aminoethoxydiphenyl borate (2-APB). Extracellular Ca(2+) blocked Na(+) currents in both gating modes with a similar inhibition constant (Ki; ~25 µM). Thus, equilibrium binding as set by the Ki of Ca(2+) blockade cannot explain the differing Ca(2+) selectivity of the two gating modes. Unlike STIM1-gated channels, Ca(2+) blockade in 2-APB-gated channels depended on the extracellular Na(+) concentration and exhibited an anomalously steep voltage dependence, consistent with enhanced Na(+) pore occupancy. Moreover, the second-order rate constants of Ca(2+) blockade were eightfold faster in 2-APB-gated channels than in STIM1-gated channels. A four-barrier, three-binding site Eyring model indicated that lowering the entry and exit energy barriers for Ca(2+) and Na(+) to simulate the faster rate constants of 2-APB-gated channels qualitatively reproduces their low Ca(2+) selectivity, suggesting that ion entry and exit rates strongly affect Ca(2+) selectivity. Noise analysis indicated that the unitary Na(+) conductance of 2-APB-gated channels is fourfold larger than that of STIM1-gated channels, but both modes of gating show a high open probability (Po; ~0.7). The increase in current noise during channel activation was consistent with stepwise recruitment of closed channels to a high Po state in both cases, suggesting that the underlying gating mechanisms are operationally similar in the two gating modes. These results suggest that both high affinity Ca(2+) binding and kinetic factors contribute to high Ca(2+) selectivity in CRAC channels.
流行的模型假设，高钙（Ca2+）选择性钙释放激活钙（CRAC）通道源于钙（Ca2+）与孔内高亲和力结合位点的紧密结合，从而阻止单价离子通量。在这里，我们研究了高亲和力 Ca（2+）结合对重组 Orai3 通道 Ca（2+）选择性的贡献，当由内质网 Ca（2+）传感器 STIM1 门控时，Orai3 通道作为高度 Ca（2+）选择性通道起作用，或者当被小分子 2-氨基乙氧基二苯硼酸盐（2-APB）激活时，作为低 Ca（2+）选择性通道起作用。细胞外 Ca（2+）以相似的抑制常数（Ki；25 µM）阻断 Na+电流。因此，由 Ca（2+）阻断的 Ki 设定的平衡结合不能解释两种门控模式的不同 Ca（2+）选择性。与 STIM1 门控通道不同，2-APB 门控通道中的 Ca（2+）阻断取决于细胞外 Na+浓度，并表现出异常陡峭的电压依赖性，这与增强的 Na+孔占有率一致。此外，2-APB 门控通道中 Ca（2+）阻断的二级速率常数比 STIM1 门控通道快八倍。四势垒、三结合位点 Eyring 模型表明，降低 Ca（2+）和 Na+的进入和退出能垒以模拟 2-APB 门控通道更快的速率常数，从质上再现其低 Ca（2+）选择性，表明离子进入和退出速率强烈影响 Ca（2+）选择性。噪声分析表明，2-APB 门控通道的单位 Na+电导比 STIM1 门控通道大四倍，但两种门控模式均显示出高开放概率（Po；0.7）。在通道激活过程中电流噪声的增加与两种情况下封闭通道逐步募集到高 Po 状态一致，这表明两种门控模式下的基本门控机制在操作上是相似的。这些结果表明，高亲和力 Ca（2+）结合和动力学因素都有助于 CRAC 通道的高 Ca（2+）选择性。
