Pore topology of the hyperpolarization-activated cyclic nucleotide-gated channel from sea urchin sperm
Pore topology of the hyperpolarization-activated cyclic nucleotide-gated channel from sea urchin sperm
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DOI：
10.1016/s0006-3495(02)73957-x
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发表时间：
2002-10-01
影响因子：
3.4
通讯作者：
Torre, V
中科院分区：
文献类型：
--
作者：
Roncaglia, P;Mistrík, P;Torre, V
电流通过超极化激活的环核苷酸门控(HCN)通道，称为i-h，在几个基本的生物过程中发挥着重要作用。推测的HCN通道孔区的序列与大多数已知的K+选择性通道的序列相似。本文用取代半胱氨酸可及性方法(SCAM)研究了海胆精子HCN通道的孔拓扑结构。野生型(W.T.)细胞内CD2+不可逆地阻断SpHCN通道。在突变体C428S中没有观察到这种阻断。胞外Cd~(2+)对W.T的I-h电流无明显抑制作用。但阻断突变通道K433C和F434C的电流。大的胞外阴离子阻断了W.T中的电流。和K433Q突变通道。这些结果表明：1)428位的半胱氨酸面向细胞内介质；2)433位的赖氨酸和434位的苯丙氨酸分别面向膜的胞外侧；3)433位的赖氨酸不介导阴离子的阻断。此外，我们的研究证实，K+通道信号序列GYG也形成了HCN通道的内孔。
The current flow through hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, referred to as I-h, plays a major role in several fundamental biological processes. The sequence of the presumed pore region of HCN channels is reminiscent of that of most known K+-selective channels. In the present work, the pore topology of an HCN channel from sea urchin sperm, called SpHCN, was investigated by means of the substituted-cysteine accessibility method (SCAM). The I-h current in the wild-type (w.t.) SpHCN channel was irreversibly blocked by intracellular Cd2+. This blockage was not observed in mutant C428S. Extracellular Cd2+ did not cause any inhibition of the I-h current in the w.t. SpHCN channel, but blocked the current in mutant channels K433C and F434C. Large extracellular anions blocked the current both in the w.t. and K433Q mutant channel. These results suggest that 1) cysteine in position 428 faces the intracellular medium; 2) lysine and phenylalanine in position 433 and 434, respectively, face the extracellular side of the membrane; and 3) lysine 433 does not mediate the anion blockade. Additionally, our study confirms that the K+ channel signature sequence GYG also forms the inner pore in HCN channels.