Holmgren M, Jurman M E, Yellen G
Department of Neurobiology, Massachusetts General Hospital, Boston 02114, USA.
J Gen Physiol. 1996 Sep;108(3):195-206. doi: 10.1085/jgp.108.3.195.
The intracellular segment of the Shaker K+ channel between transmembrane domains S4 and S5 has been proposed to form at least part of the receptor for the tethered N-type inactivation "ball." We used the approach of cysteine substitution mutagenesis and chemical modification to test the importance of this region in N-type inactivation. We studied N-type inactivation or the block by a soluble inactivation peptide ("ball peptide") before and after chemical modification by methanethiosulfonate reagents. Particularly at position 391, chemical modification altered specifically the kinetics of ball peptide binding without altering other biophysical properties of the channel. Results with reagents that attach different charged groups at 391 C suggested that there are both electrostatic and steric interactions between this site and the ball peptide. These findings identify this site to be in or near the receptor site for the inactivation ball. At many of the other positions studied, modification noticeably inhibited channel current. The accessible cysteines varied in the state-dependence of their modification, with five- to tenfold changes in reactions rate depending on the gating state of the channel.
已有人提出，Shaker钾通道跨膜结构域S4和S5之间的胞内段至少构成了与拴系式N型失活“球”相结合的受体的一部分。我们采用半胱氨酸替代诱变和化学修饰的方法来检验该区域在N型失活中的重要性。我们研究了甲硫基磺酸盐试剂进行化学修饰前后N型失活或可溶性失活肽（“球肽”）的阻断作用。特别是在391位，化学修饰特异性地改变了球肽结合的动力学，而未改变通道的其他生物物理特性。在391位半胱氨酸上连接不同电荷基团的试剂所得到的结果表明，该位点与球肽之间存在静电和空间相互作用。这些发现表明该位点位于失活球的受体位点或其附近。在研究的许多其他位置，修饰显著抑制了通道电流。可及的半胱氨酸在其修饰的状态依赖性方面存在差异，反应速率根据通道的门控状态有5至10倍的变化。
