Istituto di Biofisica, Consiglio Nazionale delle Ricerche, 16149 Genoa, Italy.
J Gen Physiol. 2012 Dec;140(6):681-96. doi: 10.1085/jgp.201210878. Epub 2012 Nov 12.
The kidney and inner ear CLC-K chloride channels, which are involved in salt absorption and endolymph production, are regulated by extracellular Ca(2+) in the millimolar concentration range. Recently, Gradogna et al. (2010. J. Gen. Physiol. http://dx.doi.org/10.1085/jgp.201010455) identified a pair of acidic residues (E261 and D278) located in the loop between helices I and J as forming a putative intersubunit Ca(2+)-binding site in hClC-Ka. In this study, we sought to explore the properties of the binding site in more detail. First, we verified that the site is conserved in hClC-Kb and rClC-K1. In addition, we could confer Ca(2+) sensitivity to the Torpedo marmorata ClC-0 channel by exchanging its I-J loop with that from ClC-Ka, demonstrating a direct role of the loop in Ca(2+) binding. Based on a structure of a bacterial CLC and a new sequence alignment, we built homology models of ClC-Ka. The models suggested additional amino acids involved in Ca(2+) binding. Testing mutants of these residues, we could restrict the range of plausible models and positively identify two more residues (E259 and E281) involved in Ca(2+) coordination. To investigate cation specificity, we applied extracellular Zn(2+), Mg(2+), Ba(2+), Sr(2+), and Mn(2+). Zn(2+) blocks ClC-Ka as well as its Ca(2+)-insensitive mutant, suggesting that Zn(2+) binds to a different site. Mg(2+) does not activate CLC-Ks, but the channels are activated by Ba(2+), Sr(2+), and Mn(2+) with a rank order of potency of Ca(2+) > Ba(2+) > Sr(2+) = Mn(2+) for the human CLC-Ks. Dose-response analysis indicates that the less potent Ba(2+) has a lower affinity rather than a lower efficacy. Interestingly, rClC-K1 shows an altered rank order (Ca(2+) > Sr(2+) >> Ba(2+)), but homology models suggest that residues outside the I-J loop are responsible for this difference. Our detailed characterization of the regulatory Ca(2+)-binding site provides a solid basis for the understanding of the physiological modulation of CLC-K channel function in the kidney and inner ear.
肾脏和内耳中的 CLC-K 氯离子通道参与盐吸收和内淋巴的产生，其功能受到细胞外钙（Ca2+）的调节，这种调节作用存在于毫摩尔浓度范围内。最近，Gradogna 等人（2010. J. Gen. Physiol. http://dx.doi.org/10.1085/jgp.201010455）发现一对位于 I 螺旋和 J 螺旋之间环上的酸性残基（E261 和 D278），构成 hClC-Ka 氯离子通道亚基间的潜在 Ca2+结合位点。在本研究中，我们试图更详细地探究该结合位点的性质。首先，我们验证了该位点在 hClC-Kb 和 rClC-K1 中是保守的。此外，我们通过交换 ClC-0 通道的 I-J 环，使其与 ClC-Ka 的 I-J 环相同，赋予了 Torpedo marmorata ClC-0 通道对 Ca2+的敏感性，表明该环在 Ca2+结合中起直接作用。基于细菌 CLC 的结构和新的序列比对，我们构建了 ClC-Ka 的同源模型。这些模型提示了另外一些参与 Ca2+结合的氨基酸残基。通过对这些残基的突变体进行测试，我们可以限制合理模型的范围，并确定另外两个参与 Ca2+配位的残基（E259 和 E281）。为了研究阳离子的特异性，我们应用了细胞外 Zn2+、Mg2+、Ba2+、Sr2+和 Mn2+。Zn2+可以阻断 ClC-Ka 及其对 Ca2+不敏感的突变体，表明 Zn2+结合在不同的位点上。Mg2+不能激活 CLC-Ks，但通道可以被 Ba2+、Sr2+和 Mn2+激活，其对人 CLC-Ks 的激活能力为 Ca2+>Ba2+>Sr2+>Mn2+。剂量反应分析表明，效力较低的 Ba2+具有较低的亲和力，而不是较低的功效。有趣的是，rClC-K1 显示出改变的顺序（Ca2+>Sr2+>>Ba2+），但同源模型表明，I-J 环外的残基是导致这种差异的原因。我们对调节性 Ca2+结合位点的详细表征，为理解肾脏和内耳中 CLC-K 通道功能的生理调节提供了坚实的基础。
