Smith-Maxwell C J, Ledwell J L, Aldrich R W
Department of Molecular and Cellular Physiology, Stanford University, Stanford, California 94305, USA.
J Gen Physiol. 1998 Mar;111(3):421-39. doi: 10.1085/jgp.111.3.421.
Substitution of the S4 of Shaw into Shaker alters cooperativity in channel activation by slowing a cooperative transition late in the activation pathway. To determine the amino acids responsible for the functional changes in Shaw S4, we created several mutants by substituting amino acids from Shaw S4 into Shaker. The S4 amino acid sequences of Shaker and Shaw S4 differ at 11 positions. Simultaneous substitution of just three noncharged residues from Shaw S4 into Shaker (V369I, I372L, S376T; ILT) reproduces the kinetic and voltage-dependent properties of Shaw S4 channel activation. These substitutions cause very small changes in the structural and chemical properties of the amino acid side chains. In contrast, substituting the positively charged basic residues in the S4 of Shaker with neutral or negative residues from the S4 of Shaw S4 does not reproduce the shallow voltage dependence or other properties of Shaw S4 opening. Macroscopic ionic currents for ILT could be fit by modifying a single set of transitions in a model for Shaker channel gating (Zagotta, W.N., T. Hoshi, and R.W. Aldrich. 1994. J. Gen. Physiol. 103:321-362). Changing the rate and voltage dependence of a final cooperative step in activation successfully reproduces the kinetic, steady state, and voltage-dependent properties of ILT ionic currents. Consistent with the model, ILT gating currents activate at negative voltages where the channel does not open and, at more positive voltages, they precede the ionic currents, confirming the existence of voltage-dependent transitions between closed states in the activation pathway. Of the three substitutions in ILT, the I372L substitution is primarily responsible for the changes in cooperativity and voltage dependence. These results suggest that noncharged residues in the S4 play a crucial role in Shaker potassium channel gating and that small steric changes in these residues can lead to large changes in cooperativity within the channel protein.
将Shaw的S4区段替换到Shaker中，会通过减缓激活途径后期的协同转变来改变通道激活的协同性。为了确定导致Shaw S4功能变化的氨基酸，我们通过将Shaw S4的氨基酸替换到Shaker中创建了几个突变体。Shaker和Shaw S4的S4氨基酸序列在11个位置上不同。仅将Shaw S4的三个不带电残基同时替换到Shaker中（V369I、I372L、S376T；ILT），就能重现Shaw S4通道激活的动力学和电压依赖性特性。这些替换导致氨基酸侧链的结构和化学性质发生非常小的变化。相比之下，用Shaw S4的中性或带负电残基替换Shaker S4中的带正电碱性残基，无法重现Shaw S4开放的浅电压依赖性或其他特性。ILT的宏观离子电流可以通过修改Shaker通道门控模型中的一组单一转变来拟合（Zagotta, W.N., T. Hoshi, and R.W. Aldrich. 1994. J. Gen. Physiol. 103:321 - 362）。改变激活中最终协同步骤的速率和电压依赖性，成功重现了ILT离子电流的动力学、稳态和电压依赖性特性。与该模型一致，ILT门控电流在通道未开放的负电压下激活，而在更正的电压下，它们先于离子电流出现，证实了激活途径中封闭状态之间存在电压依赖性转变。在ILT的三个替换中，I372L替换主要负责协同性和电压依赖性的变化。这些结果表明，S4中的不带电残基在Shaker钾通道门控中起关键作用，并且这些残基的小空间变化可导致通道蛋白内协同性的大变化。
