Ismailov I I, McDuffie J H, Benos D J
Department of Physiology and Biophysics, University of Alabama at Birmingham 35294-0005.
J Biol Chem. 1994 Apr 8;269(14):10235-41.
Purified bovine renal epithelial Na+ channels incorporated into planar lipid bilayer membranes were used to evaluate the biophysical consequences of its phosphorylation by protein kinase A (PKA). We also studied the effects of pertussis toxin-induced ADP-ribosylation on single channel activity of nonphosphorylated and PKA-phosphorylated channels. PKA-induced phosphorylation resulted in a significant increase in single channel open probability (Po) with no change in single channel conductance, as well as increased the probability of multiple channel openings in the bilayer. Further, PKA conferred a voltage sensitivity to channel gating without affecting open channel conduction properties. PKA-phosphorylated Na+ channels were inhibited by subsequent ADP-ribosylation with pertussis toxin (PTX). Addition of guanosine 5'-3-O-(thio)triphosphate reversed this inhibition. However, exposure of nonphosphorylated Na+ channels to PTX increased channel open probability by a factor of 3-5. These results demonstrate that a cAMP-dependent pathway is an important regulatory element for amiloride-sensitive Na+ channels and that the effects of PTX-induced ADP-ribosylation of the channel-associated Gi protein on function depend upon the previous phosphorylation state of the protein.
将纯化的牛肾上皮钠通道整合到平面脂质双分子层膜中，用于评估蛋白激酶A（PKA）对其磷酸化的生物物理后果。我们还研究了百日咳毒素诱导的ADP核糖基化对未磷酸化和PKA磷酸化通道单通道活性的影响。PKA诱导的磷酸化导致单通道开放概率（Po）显著增加，单通道电导无变化，同时增加了双分子层中多通道开放的概率。此外，PKA赋予通道门控电压敏感性，而不影响开放通道的传导特性。PKA磷酸化的钠通道随后被百日咳毒素（PTX）的ADP核糖基化抑制。添加鸟苷5'-3-O-（硫代）三磷酸可逆转这种抑制作用。然而，未磷酸化的钠通道暴露于PTX会使通道开放概率增加3至5倍。这些结果表明，cAMP依赖性途径是氨氯地平敏感钠通道的重要调节元件，并且PTX诱导的通道相关Gi蛋白的ADP核糖基化对功能的影响取决于该蛋白先前的磷酸化状态。
