Molecular Mechanism of NaCl Sensor in Macula Densa Cells

为了检测肾脏内向整流钾通道的细胞外钠敏感性，我们在非洲爪哇卵母细胞或表达了克隆的肾脏钾通道ROMKI的人肾细胞系HEK293上进行了电生理实验。去除胞外钠后，卵母细胞和HEK293细胞的全细胞ROMKI电流均受到明显抑制。记录在卵母细胞贴附贴片中的单通道ROMKI活性不受移液管溶液中钠的去除的影响。然而，记录在卵母细胞上的大片ROMKI电流被去钠显著抑制。Na/H逆向转运体的阻断剂阿米洛利可显著抑制去钠引起的全细胞ROMKI电流抑制。ROMKI的pH不敏感的K80M突变体对钠的去除不敏感。在两种卵母细胞…中，ROMK1与肾顶膜Na/H逆向转运体亚型共表达可增加ROMK1通道对细胞外钠的敏感性ES细胞和HEK293细胞较多。因此，我们认为ROMKI通道是由细胞外Na通过细胞内pH变化间接调节的，致密斑细胞通过特定的转运途径检测肾小管液成分的变化，并传递改变血管阻力的信号。进行膜片钳研究，以确定这些细胞的微观运输特性。切取兔肾的肾小球，切除粗大的升肢，使其完全进入致密斑区。膜片钳实验采用细胞连接(c/a)和内向外(i/o)两种方式，以直接观察MD细胞的离子通道。在c/a模式下，我们重复观察到一个20ps通道，其线性I/Y在0 mV附近反转。在I/O贴片中，电导非常相似，用氯化钠替换KCI对反转电位没有影响，但用NMDG替换所有警告时，外向电流消失。清除浴液钙(+1 mM EGTA)可使通道活动消失，这在钙离子重新注入时是可逆的。有趣的是，这个非选择性的警告通道被发现是硝苯地平敏感的，这表明它是一种钙的通道。在I/O斑块中，也发现了一个大电导阴离子通道与线性电流-电压关系(平均电导383ps)。该通道在0 mV时发生翻转，膜电位大于+30 mV时显示电压失活。去钙和添加EGTA对通道活性无影响，但可被Gd阻断。MD阴离子通道也可通透大的阴离子，包括葡萄糖酸、天冬氨酸，以及最有趣的是，ATP。100 mM钠-三磷酸腺苷可见内向外斑块的单通道事件。在相关研究中，我们使用放置在致密斑块附近的PCI2细胞的全细胞电导作为生物传感器来监测MD细胞的ATP释放。当盐浴中的氯化钠从25 mM增加到150 mM时，MD细胞有显著的ATP释放。有趣的是，在平行的细胞附着实验中，MAXI-CI通道的活性依赖于BASH[NaC l]的存在。这些结果首次证明，MD细胞具有一个对ATP具有显著通透性的Maxi-CI通道，并可能对[NaCl]的升高做出反应而释放ATP。较少

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