Ledwell J L, Aldrich R W
Department of Molecular and Cellular Physiology, Stanford University, Stanford, California 94305, USA.
J Gen Physiol. 1999 Mar;113(3):389-414. doi: 10.1085/jgp.113.3.389.
Charged residues in the S4 transmembrane segment play a key role in determining the sensitivity of voltage-gated ion channels to changes in voltage across the cell membrane. However, cooperative interactions between subunits also affect the voltage dependence of channel opening, and these interactions can be altered by making substitutions at uncharged residues in the S4 region. We have studied the activation of two mutant Shaker channels that have different S4 amino acid sequences, ILT (V369I, I372L, and S376T) and Shaw S4 (the S4 of Drosophila Shaw substituted into Shaker), and yet have very similar ionic current properties. Both mutations affect cooperativity, making a cooperative transition in the activation pathway rate limiting and shifting it to very positive voltages, but analysis of gating and ionic current recordings reveals that the ILT and Shaw S4 mutant channels have different activation pathways. Analysis of gating currents suggests that the dominant effect of the ILT mutation is to make the final cooperative transition to the open state of the channel rate limiting in an activation pathway that otherwise resembles that of Shaker. The charge movement associated with the final gating transition in ILT activation can be measured as an isolated component of charge movement in the voltage range of channel opening and accounts for 13% ( approximately 1.8 e0) of the total charge moved in the ILT activation pathway. The remainder of the ILT gating charge (87%) moves at negative voltages, where channels do not open, and confirms the presence of Shaker-like conformational changes between closed states in the activation pathway. In contrast to ILT, the activation pathway of Shaw S4 seems to involve a single cooperative charge-moving step between a closed and an open state. We cannot detect any voltage-dependent transitions between closed states for Shaw S4. Restoring basic residues that are missing in Shaw S4 (R1, R2, and K7) rescues charge movement between closed states in the activation pathway, but does not alter the voltage dependence of the rate-limiting transition in activation.
S4跨膜片段中的带电残基在决定电压门控离子通道对细胞膜两侧电压变化的敏感性方面起着关键作用。然而，亚基之间的协同相互作用也会影响通道开放的电压依赖性，并且这些相互作用可以通过在S4区域的不带电残基处进行替换而改变。我们研究了两种具有不同S4氨基酸序列的突变型Shaker通道的激活情况，即ILT（V369I、I372L和S376T）和Shaw S4（将果蝇Shaw的S4替换到Shaker中），然而它们却具有非常相似的离子电流特性。这两种突变都影响协同性，使激活途径中的协同转变成为限速步骤，并将其转移到非常正的电压，但对门控和离子电流记录的分析表明，ILT和Shaw S4突变通道具有不同的激活途径。门控电流分析表明，ILT突变的主要作用是使通道最终协同转变为开放状态成为激活途径中的限速步骤，而该激活途径在其他方面类似于Shaker通道。与ILT激活中最终门控转变相关的电荷移动可以在通道开放的电压范围内作为电荷移动的一个独立成分进行测量，并且占ILT激活途径中总电荷移动量的13%（约1.8 e0）。ILT门控电荷的其余部分（87%）在通道不开放的负电压下移动，这证实了激活途径中封闭状态之间存在类似Shaker的构象变化。与ILT相反，Shaw S4的激活途径似乎涉及封闭状态和开放状态之间的单个协同电荷移动步骤。我们无法检测到Shaw S4在封闭状态之间的任何电压依赖性转变。恢复Shaw S4中缺失的碱性残基（R1、R2和K7）可挽救激活途径中封闭状态之间的电荷移动，但不会改变激活中限速转变的电压依赖性。
