Do voltage-dependent K+ channels require Ca2+? A critical test employing a heterologous expression system.

Removal of Ca2+ from the solution bathing neurons is known in many cases to alter the gating properties of voltage-dependent K+ channels and to induce a large, nonselective "leak" conductance. We used a heterologous expression system to test whether the leak conductance observed in neurons is mediated by voltage-dependent K+ channels in an altered, debased conformation. Voltage-dependent K+ channels were expressed in an insect cell line infected with a recombinant baculovirus carrying the cDNA for Drosophila Shaker "A-type" K+ channels. These expressed channels respond to low Ca2+ identically to voltage-dependent K+ channels in native neuronal membranes; upon removal of external Ca2+, Shaker K+ currents disappear and are replaced by a steady, nonselective leak conductance. However, control cells devoid of Shaker channels were free of any voltage-dependent conductances and did not generate a leak when external Ca2+ was removed. These results show that Ca2+ is essential for proper function of voltage-dependent K+ channels and is required to stabilize the native conformations of these membrane proteins.

已有多项研究证实，将浸泡神经元的浴液中的钙离子（Ca²+）移除，在多数情况下会改变电压门控钾通道（voltage-dependent K+ channels）的门控特性，并诱导产生显著的非选择性"leak"电导。本研究采用异源表达系统，探究神经元中观测到的漏电导是否由构象异常、功能退化的电压门控钾通道所介导。我们将携带有果蝇（Drosophila）Shaker A型钾通道（Shaker "A-type" K+ channels）的互补DNA（cDNA）构建至重组杆状病毒（recombinant baculovirus）中，并用该病毒感染昆虫细胞系，以此表达电压门控钾通道。这些表达得到的通道与天然神经元膜上的电压门控钾通道对低钙环境的响应模式完全一致；当移除细胞外钙离子后，Shaker钾电流会消失，并被稳定的非选择性漏电导所取代。然而，未表达Shaker通道的对照组细胞不存在任何电压依赖性电导，且在移除细胞外钙离子后也不会产生漏电导。上述实验结果表明，钙离子（Ca2+）对电压门控钾通道的正常功能至关重要，同时也是稳定这类膜蛋白天然构象所必需的。