Sabirov R Z, Azimov R R, Ando-Akatsuka Y, Miyoshi T, Okada Y
Department of Cellular and Molecular Physiology, National Institute for Physiological Sciences, Okazaki 444-8585, Japan.
J Membr Biol. 1999 Nov 1;172(1):67-76. doi: 10.1007/s002329900584.
To examine the extracellular Na(+) sensitivity of a renal inwardly rectifying K(+) channel, we performed electrophysiological experiments on Xenopus oocytes or a human kidney cell line, HEK293, in which we had expressed the cloned renal K(+) channel, ROMK1 (Kir1. 1). When extracellular Na(+) was removed, the whole-cell ROMK1 currents were markedly suppressed in both the oocytes and HEK293 cells. Single-channel ROMK1 activities recorded in the cell-attached patch on the oocyte were not affected by removal of Na(+) from the pipette solution. However, macro-patch ROMK1 currents recorded on the oocyte were significantly suppressed by Na(+) removal from the bath solution. A blocker of Na(+)/H(+) antiporters, amiloride, largely inhibited the Na(+) removal-induced suppression of whole-cell ROMK1 currents in the oocytes. The pH-insensitive K80M mutant of ROMK1 was much less sensitive to Na(+) removal. Na(+) removal was found to induce a significant decrease in intracellular pH in the oocytes using H(+)-selective microelectrodes. Coexpression of ROMK1 with NHE3, which is a Na(+)/H(+) antiporter isoform of the kidney apical membrane, conferred increased sensitivity of ROMK1 channels to extracellular Na(+) in both the oocytes and HEK293 cells. Thus, it is concluded that the ROMK1 channel is regulated indirectly by extracellular Na(+), and that the interaction between NHE transporter and ROMK1 channel appears to be involved in the mechanism of Na(+) sensitivity of ROMK1 channel via regulating intracellular pH.
为研究肾内向整流钾通道对细胞外钠离子的敏感性，我们在非洲爪蟾卵母细胞或人肾细胞系HEK293上进行了电生理实验，在这些细胞中我们表达了克隆的肾钾通道ROMK1（Kir1.1）。当去除细胞外钠离子时，卵母细胞和HEK293细胞中的全细胞ROMK1电流均被显著抑制。在卵母细胞的细胞贴附式膜片上记录的单通道ROMK1活性不受移去吸管溶液中钠离子的影响。然而，在卵母细胞上记录的巨膜片ROMK1电流被浴液中去除钠离子显著抑制。钠离子/氢离子反向转运体的阻滞剂氨氯地平，在很大程度上抑制了卵母细胞中钠离子去除诱导的全细胞ROMK1电流的抑制。ROMK1的pH不敏感K80M突变体对钠离子去除的敏感性要低得多。使用氢离子选择性微电极发现，去除钠离子会导致卵母细胞内pH值显著降低。ROMK1与NHE3（肾顶端膜的一种钠离子/氢离子反向转运体亚型）共表达，使卵母细胞和HEK293细胞中的ROMK1通道对细胞外钠离子的敏感性增加。因此，得出结论，ROMK1通道受细胞外钠离子间接调节，并且NHE转运体与ROMK1通道之间的相互作用似乎通过调节细胞内pH值参与了ROMK1通道钠离子敏感性的机制。
