Silverman W R, Tang C Y, Mock A F, Huh K B, Papazian D M
Department of Physiology and Molecular Biology Institute, University of California Los Angeles School of Medicine, Los Angeles, California 90095-1751, USA.
J Gen Physiol. 2000 Nov;116(5):663-78. doi: 10.1085/jgp.116.5.663.
Extracellular Mg(2+) directly modulates voltage-dependent activation in ether-à-go-go (eag) potassium channels, slowing the kinetics of ionic and gating currents (Tang, C.-Y., F. Bezanilla, and D.M. Papazian. 2000. J. Gen. Physiol. 115:319-337). To exert its effect, Mg(2+) presumably binds to a site in or near the eag voltage sensor. We have tested the hypothesis that acidic residues unique to eag family members, located in transmembrane segments S2 and S3, contribute to the Mg(2+)-binding site. Two eag-specific acidic residues and three acidic residues found in the S2 and S3 segments of all voltage-dependent K(+) channels were individually mutated in Drosophila eag, mutant channels were expressed in Xenopus oocytes, and the effect of Mg(2+) on ionic current kinetics was measured using a two electrode voltage clamp. Neutralization of eag-specific residues D278 in S2 and D327 in S3 eliminated Mg(2+)-sensitivity and mimicked the slowing of activation kinetics caused by Mg(2+) binding to the wild-type channel. These results suggest that Mg(2+) modulates activation kinetics in wild-type eag by screening the negatively charged side chains of D278 and D327. Therefore, these residues are likely to coordinate the bound ion. In contrast, neutralization of the widely conserved residues D284 in S2 and D319 in S3 preserved the fast kinetics seen in wild-type eag in the absence of Mg(2+), indicating that D284 and D319 do not mediate the slowing of activation caused by Mg(2+) binding. Mutations at D284 affected the eag gating pathway, shifting the voltage dependence of Mg(2+)-sensitive, rate limiting transitions in the hyperpolarized direction. Another widely conserved residue, D274 in S2, is not required for Mg(2+) sensitivity but is in the vicinity of the binding site. We conclude that Mg(2+) binds in a water-filled pocket between S2 and S3 and thereby modulates voltage-dependent gating. The identification of this site constrains the packing of transmembrane segments in the voltage sensor of K(+) channels, and suggests a molecular mechanism by which extracellular cations modulate eag activation kinetics.
细胞外镁离子（Mg²⁺）直接调节超极化激活环核苷酸门控（eag）钾通道的电压依赖性激活，减慢离子电流和门控电流的动力学过程（Tang, C.-Y., F. Bezanilla, and D.M. Papazian. 2000. J. Gen. Physiol. 115:319 - 337）。为发挥其作用，Mg²⁺可能结合在eag电压感受器内或其附近的一个位点。我们测试了这样一个假说：位于跨膜片段S2和S3的eag家族成员特有的酸性残基，对Mg²⁺结合位点有贡献。在果蝇eag中，分别对两个eag特有的酸性残基以及在所有电压依赖性钾通道的S2和S3片段中发现的三个酸性残基进行了单个突变，将突变通道在非洲爪蟾卵母细胞中表达，并用双电极电压钳测量Mg²⁺对离子电流动力学的影响。中和S2中的eag特有的残基D278和S3中的D327消除了Mg²⁺敏感性，并模拟了Mg²⁺结合到野生型通道所导致的激活动力学减慢。这些结果表明，Mg²⁺通过屏蔽D278和D327带负电荷的侧链来调节野生型eag中的激活动力学。因此，这些残基可能与结合的离子配位。相比之下，中和S2中的广泛保守的残基D284和S3中的D319，在没有Mg²⁺的情况下保留了野生型eag中所见的快速动力学，表明D284和D319不介导Mg²⁺结合所导致的激活减慢。D284处的突变影响了eag门控途径，使Mg²⁺敏感的、限速转变的电压依赖性向超极化方向移动。另一个广泛保守的残基，S2中的D274，对Mg²⁺敏感性不是必需的，但在结合位点附近。我们得出结论，Mg²⁺结合在S2和S3之间的一个充满水的口袋中，从而调节电压依赖性门控。该位点的鉴定限制了钾通道电压感受器中跨膜片段的堆积，并提出了一种细胞外阳离子调节eag激活动力学的分子机制。
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