Distinct Mg2+-dependent steps rate limit opening and closing of a single CFTR Cl- channel
Distinct Mg2+-dependent steps rate limit opening and closing of a single CFTR Cl- channel
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DOI：
10.1085/jgp.20028594
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发表时间：
2002-06-01
影响因子：
3.8
通讯作者：
Gadsby, DC
中科院分区：
文献类型：
--
作者：
Dousmanis, AG;Nairn, AC;Gadsby, DC
ATP 结合和水解在打开和关闭囊性纤维化跨膜电导调节器 (CFTR) Cl-通道的复杂机制中所起的作用仍然存在争议。在这项工作中，通过测量暴露于 [ATP] 和 [Mg2+] 独立变化的溶液中的切除斑块中单个通道的打开和关闭速率，分别评估了 ATP 和 Mg2+ 离子对磷酸化心脏 CFTR 通道门控的贡献。发现通道开放的速率不仅受到 ATP 结合的限制，而且受到 ATP 和 Mg2+ 结合后的 Mg2+ 依赖性步骤的限制。一旦通道打开，突然撤回所有 Mg2+ 和 ATP 可能会在数十秒内阻止其关闭。但随后将这种开放通道单独暴露于 Mg2+ 离子就可以将其关闭，并且关闭率随着 [Mg2+] 在微摩尔范围内的增加而增加（与 50 muM [Mg2+] 类似，半最大值）。一个简单的解释是，通道关闭与 ATP 分子的水解在化学计量上耦合，尽管连续清洗，该分子仍与开放的 CFTR 通道紧密相关。如果正确的话，该 ATP 分子似乎能够在控制通道关闭的催化位点中驻留超过一分钟，这意味着即使没有 Mg2+ 离子，该位点也必须捕获 ATP，或者对 ATP 具有内在的高表观亲和力。这种通过 ATP 分子的紧密结合或封闭来稳定 CFTR 的开放通道构象，与通过 GTP 来稳定 G 蛋白的活性构象相呼应。
The roles played by ATP binding and hydrolysis in the complex mechanisms that open and close cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels remain controversial. In this work, the contributions made by ATP and Mg2+ ions to the gating of phosphorylated cardiac CFTR channels were evaluated separately by measuring the rates of opening and closing of single channels in excised patches exposed to solutions in which [ATP] and [Mg2+] were varied independently. Channel opening was found to be rate-limited not by the binding of ATP alone, but by a Mg2+-dependent step that followed binding of both ATP and Mg2+. Once a channel had opened, sudden withdrawal of all Mg2+ and ATP could prevent it from closing for tens of seconds. But subsequent exposure of such an open channel to Mg2+ ions alone could close it, and the closing rate increased with [Mg2+] over the micromolar range (half maximal at similar to50 muM [Mg2+]). A simple interpretation is that channel closing is stoichiometrically coupled to hydrolysis of an ATP molecule that remains tightly associated with the open CFTR channel despite continuous washing. If correct, that ATP molecule appears able to reside for over a minute in the catalytic site that controls channel closing, implying that the site must entrap, or have an intrinsically high apparent affinity for, ATP, even without a Mg2+ ion. Such stabilization of the open-channel conformation of CFTR by tight binding, or occlusion, of an ATP molecule echoes the stabilization of the active conformation of a G protein by GTP.