Regulation and expression of ion channels in the renal tubule cells

沿着肾单位的肾钾通道在膜电位的形成中起关键作用，膜电位是小管上皮流变离子运输的驱动力。在这项研究中，我们采用膜片钳技术研究了负鼠和人肾近端小管细胞中K^+通道的调节机制。在负鼠肾细胞中，在对照条件下，细胞贴附斑块中最常观察到向内整流的K^+通道，其向内电导约为90pS。我们发现蛋白激酶A （PKA）增强了K^+通道的活性，而蛋白磷酸酶1型和2A型（PP-1和PP-2A）抑制了K^+通道的活性。这些结果表明pka介导的磷酸化至少部分被该通道中的PP-1和PP-2A去磷酸化。另一方面，先前的报道表明，ATP对近端小管细胞内纠偏K^+通道的影响在不同动物物种中有所不同。也就是说，一个是atp敏感（inh…More ibitable）通道，另一个是atp依赖通道。然而，对人类近端小管细胞中的K^+通道知之甚少。因此，下一步，我们将膜片钳技术应用于正常肾源的人近端小管细胞。我们在近端小管细胞表面膜上发现了一个向内整流的K^+通道，其向内电导约为42pS。这个K^+通道是细胞附着斑块中最主要的K^+通道，并且需要ATP来维持内向外斑块中的通道活性。ATP对通道活性的影响呈剂量依赖性刺激。因此，人近端小管细胞的内整流K^+通道不是atp敏感通道。此外，我们发现PKA增强了通道活性，并受到内部ph的显著影响，除此之外，我们还发现了一个大电导的K^+通道，该通道被Ca^<2+>激活，并被ATP抑制。我们的数据表明，人类近端小管细胞对离子通道的调节机制和生理功能的研究是有用的。少

Renal potassium channels along the nephron play a key role in formation of membrane potential, which serves as driving forces for rheogenic ion transport across the tubular epithelia. In this study, we investigated the regulatory mechanisms of K^+ channels in opossum and human renal proximal tubule cells by using the patch-clamp technique. In opossum kidney cells, inwardly rectifying K^+ channels with inward conductance of about 90pS were most frequently observed in cell-attached patches under the control condition. We found out that activity of this K^+ channel was enhanced by protein kinase A (PKA) and inhibited by protein phosphatase types 1 and 2A (PP-1 and PP-2A). These results suggest that PKA-mediated phosphorylation is dephosphorylated at least in part by PP-1 and PP-2A in this channel.On the other hand, previous reports showed that ATP effects on inwardly rectifying K^+ channels in proximal tubule cells varied in individual animal species. Namely, one is the ATP-sensitive (inh … More ibitable) channel and the other is the ATP-dependent channel. However, little is known about K^+ channels in human proximal tubule cells. Thus in the next step, we applied the patch-clamp technique to the human proximal tubule cells of normal kidney origin. We identified an inwardly rectifying K^+ channels with an inward conductance of about 42pS in the surface membrane of the proximal tubule cells. This K^+ channel was the most dominant K^+ channel in cell-attached patches, and ATP was required to maintain channel activity in inside-out patches. The ATP effect on channel activity was dose-dependently stimulatory. Thus the inwardly rectifying K^+ channel in the human proximal tubule cells was not ATP-sensitive channel. Moreover, we found that channel activity was enhanced by PKA, and significantly affected by internal pH.In addition to this channel, we characterized a large conductance K^+ channel, which is activated by Ca^<2+> and inhibited by ATP.Our data suggest that the human proximal tubule cells are useful for investigation of regulatory mechanisms and physiological functions of ion channels. Less