Molecular Mechanism of NaCl Sensor in Macula Densa Cells

为了检测肾内纠偏K通道的细胞外钠敏感性，我们对爪蟾卵母细胞和人肾细胞系HEK293进行了电生理实验，其中我们表达了克隆的肾K通道ROMKI。当细胞外Na被去除时，卵母细胞和HEK293细胞的全细胞ROMKI电流均被明显抑制。单通道ROMKI活性记录在卵母细胞附着的贴片上，不受移液液中去除Na的影响。然而，在卵母细胞上记录的大补丁ROMKI电流被Na去除显著抑制。一种Na/H反转运蛋白的黑剂，阿米洛利，在很大程度上抑制了Na去除诱导的全细胞ROMKI电流的抑制。ROMKI对ph不敏感的K80M突变体对Na去除的敏感性要低得多。在卵母细胞和HEK293细胞中，ROMK1与肾顶膜的Na/H反转运异构体的共表达增加了ROMKI通道对细胞外Na的敏感性。因此，我们得出结论，ROMKI通道是通过细胞内pH变化间接受到细胞外Na的调节。黄斑致密（MD）细胞通过特定的运输途径检测管状液体成分的变化，并传递改变血管阻力的信号。膜片钳研究用于确定这些细胞的微观运输特性。从兔肾中剥离肾小球，切除粗大的升肢，以完全接近黄斑致密区。膜片钳实验采用细胞贴壁（c/a）或内外（I/o）两种构型，直接观察MD细胞中的离子通道。在c/a模式下，我们反复观察到一个20 pS的通道，其线性I/Y在0 mV附近反转。在I/o片中，电导非常相似，用NaCl代替KCI对反转电位没有影响，但用NMDG代替所有警告液后，向外电流消失。消除浴钙（+ 1mM EGTA）会消除通道活性，这在钙的再读时是可逆的。有趣的是，这种非选择性谨慎通道被发现对硝苯地平敏感，这表明它是钙的途径。在I/o贴片中，还发现了一个具有线性电流电压关系（平均电导383 pS）的大电导阴离子通道。当膜电位大于+30 mV时，该通道在0 mV时反转，显示电压失活。通道活性不受去除钙和加入EGTA的影响，但被钆阻断。MD阴离子通道也可渗透到大阴离子，包括葡萄糖酸盐、天冬氨酸和最有趣的ATP。在100 mM Na-ATP中发现了由内向外的单通道事件。在相关研究中，我们将PCI2细胞的全细胞电导放置在靠近黄斑致密斑块的位置，作为生物传感器来监测MD细胞释放ATP。当培养液NaCl浓度从25 mM增加到150 mM时，MD细胞中有显著的ATP释放。有趣的是，在平行细胞附着实验中，最大ci通道活性依赖于浴液[NaCl]的存在。这些结果首次表明，MD细胞具有一个最大ci通道，该通道对ATP具有显著的渗透性，并可能随着[NaCl]的增加而释放ATP。少

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, ROMKI. When extracellular Na was removed, the whole-cell ROMKI currents were markedly suppressed in both the oocytes and HEK293 cells. Single-channel ROMKI activities recorded in the cell-attached patch on the oocyte were not affected by removal of Na from the pipette solution. However, macro-patch ROMKI currents recorded on the oocyte were significantly suppressed by Na removal. A blacker of Na/H antiporters, amiloride, largely inhibited the Na removal-induced suppression of whole-cell ROMKI currents. The pH-insensitive K80M mutant of ROMKI was much less sensitive to Na removal. Coexpression of ROMK1 with a Na/H antiporter isoform of the kidney apical membrane, conferred increased sensitivity of ROMKI channels to extracellular Na in both the oocyt … More es and HEK293 cells. Thus, it is concluded that the ROMKI channel is regulated indirectly by extracellular Na via intracellular pH changes.Macula densa (MD) cells detect changes in tubular fluid composition through specific transport pathways and transmit signals which alter vascular resistance. Patch clamp studies were performed to define microscopic transport properties of these cells. Glomeruli were dissected from rabbit kidney and thick ascending limb removed to gain complete access to the macula densa. Patch clamp experiments in cell-attached (c/a) or inside- out (I/o) configurations were performed to directly observe ionic channels in MD cells. In c/a mode, we repeatedly observed a 20 pS channel with a linear I/Y which reversed near 0 mV. In I/o patches, the conductance was very similar, and the reversal potential was unaffected by replacing KCI with NaCl but outward currents disappeared upon bath replacement of all cautions with NMDG. Elimination of bath calcium (+ 1mM EGTA) abolished channel activity, and this was reversible upon readdition of calcium. Interestingly, this non-selective caution channel was found to be nifedipine-sensitive which suggests that it serve as a pathway for calcium.In I/o patches, a large conductance anion channel was also identified with a linear current voltage relationship (mean conductance 383 pS). This channel reversed at 0 mV and displayed voltage inactivation for membrane potentials more positive than +30 mV. Channel activity was unaffected by removal of calcium and addition of EGTA but was blocked by gadolinium. The MD anion channel was also permeable to large anions including gluconate, aspartate, and, most interestingly, ATP. Single channel events in inside-out patches was found with 100 mM Na-ATP. In related studies, we used whole-cell conductance of PCI2 cells, placed close to the macula densa plaque, as a biosensor to monitor ATP release by MD cells. In response to an increase in bath NaCl from 25 to 150 mM, there was a significant release of ATP from MD cells. Interestingly, in parallel cell-attached experiments, maxi-CI channel activity was dependent upon the presence of bath [NaCl]. These results demonstrate, for the first time, that MD cells possess a maxi-CI channel which exhibits significant permeability to ATP and may release ATP in response to increases in [NaCl]. Less