Elinder Fredrik, Madeja Michael, Zeberg Hugo, Århem Peter
Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden.
Institute for Physiology, University of Münster and Hertie Research Group at Center for Physiology, University of Frankfurt, Germany.
Biophys J. 2016 Oct 18;111(8):1679-1691. doi: 10.1016/j.bpj.2016.08.043.
The transmembrane voltage needed to open different voltage-gated K (Kv) channels differs by up to 50 mV from each other. In this study we test the hypothesis that the channels' voltage dependences to a large extent are set by charged amino-acid residues of the extracellular linkers of the Kv channels, which electrostatically affect the charged amino-acid residues of the voltage sensor S4. Extracellular cations shift the conductance-versus-voltage curve, G(V), by interfering with these extracellular charges. We have explored these issues by analyzing the effects of the divalent strontium ion (Sr) on the voltage dependence of the G(V) curves of wild-type and chimeric Kv channels expressed in Xenopus oocytes, using the voltage-clamp technique. Out of seven Kv channels, Kv1.2 was found to be most sensitive to Sr (50 mM shifted G(V) by +21.7 mV), and Kv2.1 to be the least sensitive (+7.8 mV). Experiments on 25 chimeras, constructed from Kv1.2 and Kv2.1, showed that the large Sr-induced G(V) shift of Kv1.2 can be transferred to Kv2.1 by exchanging the extracellular linker between S3 and S4 (L3/4) in combination with either the extracellular linker between S5 and the pore (L5/P) or that between the pore and S6 (LP/6). The effects of the linker substitutions were nonadditive, suggesting specific structural interactions. The free energy of these interactions was ∼20 kJ/mol, suggesting involvement of hydrophobic interactions and/or hydrogen bonds. Using principles from double-layer theory we derived an approximate linear equation (relating the voltage shifts to altered ionic strength), which proved to well match experimental data, suggesting that Sr acts on these channels mainly by screening surface charges. Taken together, these results highlight the extracellular surface potential at the voltage sensor as an important determinant of the channels' voltage dependence, making the extracellular linkers essential targets for evolutionary selection.
打开不同的电压门控钾离子（Kv）通道所需的跨膜电压彼此相差可达50毫伏。在本研究中，我们检验了这样一个假设：通道的电压依赖性在很大程度上由Kv通道细胞外环的带电氨基酸残基设定，这些残基通过静电作用影响电压感受器S4的带电氨基酸残基。细胞外阳离子通过干扰这些细胞外电荷来移动电导-电压曲线G(V)。我们使用电压钳技术，通过分析二价锶离子（Sr）对非洲爪蟾卵母细胞中表达的野生型和嵌合Kv通道G(V)曲线电压依赖性的影响，来探究这些问题。在七个Kv通道中，发现Kv1.2对Sr最敏感（50 mM使G(V)正向移动21.7毫伏），而Kv2.1最不敏感（正向移动7.8毫伏）。对由Kv1.2和Kv2.1构建的25个嵌合体进行的实验表明，通过交换S3和S4之间的细胞外环（L3/4），并结合S5和孔之间的细胞外环（L5/P）或孔和S6之间的细胞外环（LP/6），Kv1.2中由Sr诱导的较大G(V)位移可以转移到Kv2.1。连接子替换的效应是非加性的，表明存在特定的结构相互作用。这些相互作用的自由能约为20 kJ/mol，表明涉及疏水相互作用和/或氢键。利用双层理论的原理，我们推导了一个近似线性方程（将电压位移与离子强度变化相关联），该方程被证明与实验数据匹配良好，表明Sr主要通过屏蔽表面电荷作用于这些通道。综上所述，这些结果突出了电压感受器处的细胞外表面电位是通道电压依赖性的重要决定因素，使得细胞外环成为进化选择的关键靶点。
