Amasheh S, Weber W
Institute for Clinical Physiology, Freie Universität Berlin, UKBF, Hindenburgdamm 30, D-12200 Berlin, Germany.
J Membr Biol. 1999 Nov 15;172(2):169-79. doi: 10.1007/s002329900594.
Removal of extracellular Ca(2+) activates ion channels in the plasma membrane of defolliculated oocytes of the South Africa clawed toad Xenopus laevis. At present, there is controversy about the nature of the Ca(2+)-inactivated ion channels. Recently, we identified one of these channels as a Ca(2+)-inactivated Cl(-) channel (CaIC) using single channel analysis. In this work we confirm and extend previous observations on the CaIC by presenting a decisive extension of the regulation and inhibition profile. CaIC current is reversibly blocked by the divalent and trivalent cations Zn(2+) (half-maximal blocker concentration, K(1/2) = 8 muM), and Gd(3+) (K(1/2) = 20 muM). Furthermore, CaIC is inhibited by the specific Cl(-) channel blocker NPPB (K(1/2) approximately 3 muM). Interestingly, CaIC-mediated currents are further sensitive to the cation channel inhibitor amiloride (500 muM) but insensitive to its high affinity analogue benzamil (100 muM). An investigation of the pH-dependence of the CaIC revealed a reduction of currents in the acidic range. Using simultaneous measurements of membrane current (I(m)), conductance (G(m)) and capacitance (C(m)) we demonstrate that Ca(2+) removal leads to instant activation of CaIC already present in the plasma membrane. Since C(m) remains constant upon Ca(2+) depletion while I(m) and G(m) increase drastically, no exocytotic transport of CaIC from intracellular pools and functional insertion into the plasma membrane is involved in the large CaIC currents. A detailed overview of applicable blockers is given. These blockers are useful when oocytes are utilized as an expression system for foreign proteins whose investigations require Ca(2+)-free solutions and disturbances by CaIC currents are unwanted. We further compare and discuss our results with data of Ca(2+)-inactivated cation channels reported by other groups.
去除细胞外的Ca(2+)可激活南非爪蟾非洲爪蟾去滤泡卵母细胞质膜中的离子通道。目前，关于Ca(2+)失活离子通道的性质存在争议。最近，我们通过单通道分析将其中一种通道鉴定为Ca(2+)失活的Cl(-)通道（CaIC）。在这项工作中，我们通过展示调节和抑制谱的决定性扩展来证实并扩展先前对CaIC的观察。CaIC电流可被二价和三价阳离子Zn(2+)（半数最大阻断浓度，K(1/2)=8μM）和Gd(3+)（K(1/2)=20μM）可逆阻断。此外，CaIC受到特异性Cl(-)通道阻断剂NPPB（K(1/2)约为3μM）的抑制。有趣的是，CaIC介导的电流对阳离子通道抑制剂阿米洛利（500μM）更敏感，但对其高亲和力类似物苯扎米（100μM）不敏感。对CaIC的pH依赖性研究表明，在酸性范围内电流会降低。通过同时测量膜电流（I(m)）、电导（G(m)）和电容（C(m)），我们证明去除Ca(2+)会导致质膜中已存在的CaIC立即激活。由于Ca(2+)耗竭时C(m)保持恒定，而I(m)和G(m)急剧增加，因此大的CaIC电流不涉及CaIC从细胞内池的胞吐转运和功能性插入质膜。给出了适用阻断剂的详细概述。当卵母细胞用作外源蛋白的表达系统时，这些阻断剂很有用，因为对外源蛋白的研究需要无Ca(2+)溶液且不需要CaIC电流的干扰。我们还将我们的结果与其他组报道的Ca(2+)失活阳离子通道的数据进行了比较和讨论。
