Molecular Mechanism of NaCl Sensor in Macula Densa Cells

为了研究肾内向整流K通道的细胞外Na敏感性，我们对非洲爪蟾卵母细胞或人肾细胞系HEK 293进行了电生理学实验，其中我们表达了克隆的肾K通道ROMKI。当细胞外Na被去除时，在卵母细胞和HEK 293细胞中的全细胞ROMKI电流均被显著抑制。单通道ROMKI活动记录在卵母细胞上的细胞贴附补丁不受影响，从移液器溶液中去除Na。然而，记录在卵母细胞上的宏斑ROMKI电流被Na去除显著抑制。一个黑色的Na/H反向转运蛋白，阿米洛利，在很大程度上抑制了Na去除诱导的抑制全细胞ROMKI电流。ROMKI的pH不敏感的K80 M突变体对Na去除的敏感性低得多。ROMK 1与肾顶膜Na/H逆向转运蛋白亚型的共表达，增加了ROMKI通道对卵母细胞和卵母细胞中细胞外Na+的敏感性。 关于我们 es和HEK 293细胞。因此，可以得出结论，ROMKI通道是通过细胞内pH值的变化间接调节细胞外Na。致密斑（MD）细胞通过特定的运输途径检测肾小管液组成的变化，并传递信号，改变血管阻力。进行膜片钳研究以确定这些细胞的微观转运特性。从兔肾中解剖肾小球，并切除厚的升支以完全进入致密斑。在细胞贴附（c/a）或由内而外（I/o）构型中进行膜片钳实验以直接观察MD细胞中的离子通道。在c/a模式下，我们反复观察到具有线性I/Y的20 pS通道，其在0 mV附近反转。在I/O斑片中，电导非常相似，并且用NaCl代替KCl不影响反转电位，但用NMDG代替所有注意事项后，外向电流消失。消除浴钙（+1 mM EGTA）废除通道活动，这是可逆的钙后，再补充。有趣的是，这种非选择性谨慎通道被发现是硝苯地平敏感的，这表明它作为一个途径的Ca 2+。在I/O patches，一个大电导阴离子通道也被确定为具有线性电流-电压关系（平均电导383 pS）。该通道在0 mV时逆转，并显示膜电位比+30 mV更正的电压失活。通道活性不受钙的去除和EGTA的加入，但被钆阻断。MD阴离子通道也可渗透大的阴离子，包括葡萄糖酸盐，天冬氨酸，最有趣的是，ATP。在100 mM Na-ATP中发现了由内而外贴片中的单通道事件。在相关研究中，我们使用PCI 2细胞的全细胞电导，放置在致密斑斑块附近，作为生物传感器来监测MD细胞释放ATP。在响应增加浴NaCl从25至150 mM，有一个显着的释放ATP从MD细胞。有趣的是，在平行的细胞附着实验中，maxi-Cl通道活性取决于浴[NaCl]的存在。这些结果首次证明，MD细胞具有对ATP表现出显著渗透性的maxi-CI通道，并且可以响应于[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