Schindl Rainer, Bergsmann Judith, Frischauf Irene, Derler Isabella, Fahrner Marc, Muik Martin, Fritsch Reinhard, Groschner Klaus, Romanin Christoph
Institute of Biophysics, University of Linz, Linz, Austria.
J Biol Chem. 2008 Jul 18;283(29):20261-7. doi: 10.1074/jbc.M803101200. Epub 2008 May 21.
Stim1 in the endoplasmic reticulum and the three Orai (also termed CRACM) channels in the plasma-membrane are main components of native Ca(2+) release-activated Ca(2+) channels. A pharmacological hallmark of these channels is their distinct sensitivity to 2-aminoethoxydiphenyl borate (2-APB). Here we report that Orai3 currents can be robustly stimulated by 75 microm 2-APB independent of Stim1, whereas 2-APB at similar concentrations inhibited store-operated Orai1 currents. 2-APB did not only promote currents through Orai3 channels but also dramatically altered ion selectivity of Orai3 channels. This allowed for permeation of monovalent cations both in the inward as well as outward direction, which is in sharp contrast to the high Ca(2+) selectivity of store-operated Orai3 currents. An Orai3-R66W mutant, which lacked in analogy to the severe combined immune deficiency mutant Orai1-R91W store-operated activation, was also found to be resistant to 2-APB stimulation. The change in selectivity by 2-APB was associated with an increase in Orai3 minimum pore size from about 3.8A to more than 5.34 A. In line with a potential interaction of 2-APB with the Orai3 pore, among three pore mutants tested, the Orai3 E165Q mutant particularly resembled in its permeation properties those of 2-APB stimulated Orai3 and additionally exhibited a reduced response to 2-APB. In aggregate, stimulation of Orai3 currents by 2-APB occurred along with an alteration of the permeation pathway that represents a unique mechanism for regulating ion channel selectivity by chemical compounds.
内质网中的 Stim1 和质膜中的三种 Orai（也称为 CRACM）通道是天然钙释放激活钙通道的主要成分。这些通道的一个药理学特征是它们对 2-氨基乙氧基二苯基硼酸盐（2-APB）具有独特的敏感性。在此我们报告，75 微摩尔的 2-APB 可强烈刺激 Orai3 电流，且与 Stim1 无关，而类似浓度的 2-APB 会抑制储存操纵的 Orai1 电流。2-APB 不仅促进通过 Orai3 通道的电流，还显著改变了 Orai3 通道的离子选择性。这使得单价阳离子能够向内和向外通透，这与储存操纵的 Orai3 电流的高钙选择性形成鲜明对比。与严重联合免疫缺陷突变体 Orai1-R91W 缺乏储存操纵激活类似，Orai3-R66W 突变体也被发现对 2-APB 刺激具有抗性。2-APB 引起的选择性变化与 Orai3 最小孔径从约 3.8 埃增加到超过 5.34 埃有关。与 2-APB 与 Orai3 孔的潜在相互作用一致，在所测试的三个孔突变体中，Orai3 E165Q 突变体在其通透特性上特别类似于 2-APB 刺激的 Orai3，并且对 2-APB 的反应也降低。总体而言，2-APB 对 Orai3 电流的刺激伴随着通透途径的改变，这代表了一种通过化合物调节离子通道选择性的独特机制。
