Molecular Mechanisms for Regulation of Renal Ion Channels

应用膜片钳技术研究了近端小管细胞基底膜K^+通道的调控机制及其生理意义。在牛蛙肾中，在离体近端小管细胞的基底外侧膜上存在向内矫正的K^+通道，其向内电导约为50ps。细胞内碱化可增强K^+通道的活性，而H^+离子载体（FCCP）存在时，K^+通道的酸化可抑制K^+通道的活性。该通道也受细胞内ATP的调节，并在近端小管细胞基底外侧膜电位的形成中起主要作用。在负鼠肾近端小管（OKP）细胞中，观察到向内传导约90 pS的K^+通道，该通道也被报道为ph敏感并受ATP调节。K^+通道的活性被蛋白激酶抑制剂抑制，并通过在细胞贴附斑块中应用二丁基- camp或二丁基- cgmip增强。在ATP存在的情况下，直接应用cAMP-或cgmp依赖性蛋白激酶（PKA或PKG）可增强内向外斑块的通道活性（3mm）。相反，在Ca^<2+> (1muM)存在的情况下，直接应用蛋白激酶C （PKC）可以抑制K^+通道的活性。这些结果表明，OKP细胞中的K^+通道至少部分受到两种不同磷酸化过程的调节，PKA或pkg介导的磷酸化和pkc介导的磷酸化。此外，我们的研究结果表明，心房利钠肽是刺激pkg介导的OKP细胞磷酸化的激动剂之一。

The regulatory mechanism and physiological significance of K^+ channels in the basolateral membrane of proximal tubule cells have been investigated by using the patch-clamp technique. In bullfrog kidneys, inwardly rectifying K^+ channels with the inward conductance of about 50 pS are present in the basolateral membrane of isolated proximal tubule tubule cells. Activity of the K^+ channel is enhanced by intracellular alkalinization, and is inhibited by its acidification in cell-attached patches in the presence of H^+ ionophore, FCCP.The channel is regulated also by intracellular ATP, and plays a major role in formation of the basolateral membrane potential of the proximal tubule cell.In opossum kidney proximal tubule (OKP) cells, the K^+ channel with inward conductance of about 90 pS was observed, which was also reported to be pH-sensitive and regulated by ATP.Activity of the K^+ channel is inhibited by protein kinase inhibitors, and is enhanced by application of dibutyryl-cAMP or dibutyryl-cGMiP in cell-attached patches. Direct application of cAMP- or cGMP-dependent protein kinase (PKA or PKG) enhanced channel activity in inside-out patches in the presence of ATP (3 mM). In contrast, activity of the K^+ channel is inhibited by direct application of protein kinase C (PKC) in the presence of Ca^<2+> (1muM).These results suggest that the K^+ channel in OKP cells is regulated at least in part by two different phosphorylation processes, PKA or PKG-mediated phosphorylation and PKC-mediated phosphorylation. Moreover, our results suggest that atrial natriuretic peptide is one of the agonist which stimulates PKG-mediated phosphorylation in OKP cells.