平面脂双层中重组的肾上皮钠通道受钠离子和钙离子的调节。
Regulation by Na+ and Ca2+ of renal epithelial Na+ channels reconstituted into planar lipid bilayers.
作者信息
Ismailov I I, Berdiev B K, Benos D J
机构信息
Department of Physiology and Biophysics, University of Alabama at Birmingham 35294, USA.
出版信息
J Gen Physiol. 1995 Sep;106(3):445-66. doi: 10.1085/jgp.106.3.445.
Purified bovine renal epithelial Na+ channels when reconstituted into planar lipid bilayers displayed a specific orientation when the membrane was clamped to -40 mV (cis-side) during incorporation. The trans-facing portion of the channel was extracellular (i.e., amiloride-sensitive), whereas the cis-facing side was intracellular (i.e., protein kinase A-sensitive). Single channels had a main state unitary conductance of 40 pS and displayed two subconductive states each of 12-13 pS, or one of 12-13 pS and the second of 24-26 pS. Elevation of the [Na+] gradient from the trans-side increased single-channel open probability (Po) only when the cis-side was bathed with a solution containing low [Na+] (< 30 mM) and 10-100 microM [Ca2+]. Under these conditions, Po saturated with increasing [Na+]trans. Buffering of the cis compartment [Ca2+] to nearly zero (< 1 nM) with 10 mM EGTA increased the initial level of channel activity (Po = 0.12 +/- 0.02 vs 0.02 +/- 0.01 in control), but markedly reduced the influence of both cis- and trans-[Na+] on Po. Elevating [Ca2+]cis at constant [Na+] resulted in inhibition of channel activity with an apparent [KiCa2+] of 10-100 microM. Protein kinase C-induced phosphorylation shifted the dependence of channel Po on [Ca2+]cis to 1-3 microM at stationary [Na+]. The direct modulation of single-channel Po by Na+ and Ca2+ demonstrates that the gating of amiloride-sensitive Na2+ channels is indeed dependent upon the specific ionic environment surrounding the channels.
纯化的牛肾上皮钠通道在重构到平面脂质双分子层中时，当在整合过程中将膜钳制在-40 mV（顺式侧）时会呈现特定的取向。通道的反式面对部分是细胞外的（即对氨氯地平敏感），而顺式面对侧是细胞内的（即对蛋白激酶A敏感）。单通道的主要状态单位电导为40 pS，并呈现出两个亚电导状态，每个亚电导状态为12 - 13 pS，或者一个为12 - 13 pS，另一个为24 - 26 pS。仅当顺式侧用含有低[Na⁺]（< 30 mM）和10 - 100 μM [Ca²⁺]的溶液灌注时，反式侧[Na⁺]梯度的升高才会增加单通道开放概率（Po）。在这些条件下，Po随[Na⁺]反式的增加而饱和。用10 mM EGTA将顺式隔室[Ca²⁺]缓冲至接近零（< 1 nM）会增加通道活性的初始水平（Po = 0.12 ± 0.02，而对照中为0.02 ± 0.01），但显著降低了顺式和反式[Na⁺]对Po的影响。在恒定[Na⁺]下升高[Ca²⁺]顺式会导致通道活性受到抑制，表观[KiCa²⁺]为10 - 100 μM。蛋白激酶C诱导的磷酸化在固定[Na⁺]时将通道Po对[Ca²⁺]顺式的依赖性转变为1 - 3 μM。Na⁺和Ca²⁺对单通道Po的直接调节表明，氨氯地平敏感的Na⁺通道的门控确实取决于通道周围的特定离子环境。
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