Regulation and expression of ion channels in the renal tubule cells

肾脏沿肾单位的钾通道在膜电位的形成中起关键作用，膜电位是肾小管上皮细胞跨肾小管上皮细胞流变性离子转运的驱动力。本研究利用膜片钳技术研究了负鼠和人肾小管上皮细胞K~(++)通道的调节机制。在负鼠肾细胞中，最常见的内向整流性K~(++)通道出现在对照条件下的细胞贴壁上。我们发现该K~+通道的活性被蛋白激酶A(PKA)增强，而被蛋白磷酸酶1和2A型(PP-1和PP-2A)抑制。这些结果表明，PKA介导的磷酸化至少部分地被PP-1和PP-2A去磷酸化。另一方面，先前的报道表明，ATP对近端小管细胞内向整流钾离子通道的作用在不同的动物物种中是不同的。也就是说，一种是三磷酸腺苷敏感(InH…更具可比特)通道，另一种是依赖于ATP的通道。然而，对人近端肾小管上皮细胞的K~(++)通道知之甚少。因此，在下一步，我们将膜片钳技术应用于正常肾来源的人近端小管细胞。我们在近端小管细胞的表面膜上发现了一个内向整流性K~(++)通道，内向电导约为42pS。这种K^+通道是细胞贴附斑块中最主要的K^+通道，需要ATP来维持内向外斑块的通道活性。ATP对通道活动的影响具有剂量依赖性。因此，人近端肾小管上皮细胞的内向整流性K~+通道不是ATP敏感通道。此外，我们还发现PKA能增强通道的活性，而内源性PKA对通道的活性有显著的影响。此外，我们还研究了一个大电导K~(++)通道，该通道被Ca~(2+)激活，被ATP抑制。我们的数据表明，人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。较少

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