Regulation of rat epithelial NaィイD1+ィエD1 channel through its nucleotide binding domain

表观Na D1+表观D1通道（ENaC），由3个subunits （αβγ）组成，在不同的表观D1-表观D1吸收表观，并在盐和水平衡中发挥关键作用，并在血液压力的调节中发挥作用。使用贴片钳技术，我现在已经研究了细胞质ATP对大鼠αβγENaC （renaC）活动的影响，并在NIH-3 T3细胞中稳定地表现出来。生物物理性质的两种宏观和微观淀粉样物质--敏感电流的RENaC在这些细胞中是相似的，这些细胞在Xenopus oocytes和MDCK细胞中也是如此。在传统的全细胞贴片钳记录中，细胞与富含Cs-谷氨酸盐的管溶液的分化导致没有ATP导致向内全细胞当前属性对renaC活动的运行下降，在5分钟内减少约50%。该运行是由ATP的内部应用（± 2 mM）抑制的，不依赖于Mgy D12+ Ye D1在管盒溶液中的存在。两者都是非氢化ATP模拟， AMP-PNP，和不可能的氢化模拟，ATP-γS，在2 mM，在预防运行时也很有效，同时GTP， UTP。ADP or AMP （at 2 mM） were relatively ineffective， with an apparent order of effectiveness of ATP > ADP = GTP > UTP =AMP。在单一通道级别上，单元renaC通道外补丁中的通道行为不受细胞质ATP、Mgy D12+ y D1浓缩或替代各种核otides的影响。通道活动（NPo），定义为通道（N）及其开放概率（Po）的数量，在细胞质ATP的体外补丁中也降低，而这种降低被细胞质ATP （2 mM）明显抑制，但不是ADP。这些结果提供了一种新颖的对RENaC活动的指导，很可能通过细胞质ATP的非氢化绑定。

The epithelial NaィイD1+ィエD1 channel (ENaC), composed of 3 subunits (αβγ), is expressed in various NaィイD1-ィエD1 absorbing epithelia and plays a critical role in salt and water balance and in the regulation of blood pressure. Using patch-clamp techniques, I have now examined the effect of cytosolic ATP on the activity of the rat αβγENaC (rENaC) stably expressed in NIH-3T3 cells. Biophysical properties of both macroscopic and microscopic amiloride-sensitive currents of rENaC in these cells were similar to those heterologously expressed in Xenopus oocytes and in MDCK cells. In conventional whole-cell patch clamp recordings, dialysis of the cells with a Cs-glutamate-rich pipette solution containing no ATP caused a run-down of the inward whole-cell current attributable to rENaC activity, declining by about 50% within 5 min. The rundown was inhibited by intracellular application of ATP (≧2mM), an inhibition not dependent on the presence of MgィイD12+ィエD1 in the pipette solution. Both the nonhydrolyzable ATP analogue, AMP-PNP, and the poorly hydrolyzable analogue, ATP-γS, at 2 mM, were also effective at preventing the run-down, while GTP, UTP. ADP or AMP (at 2mM) were relatively ineffective, with an apparent order of effectiveness of ATP > ADP = GTP > UTP =AMP. At the single channel level, unitary rENaC channel conductance in outside-out patches was not affected by cytosolic ATP, MgィイD12+ィエD1 concentrations or substitution with various nucleotides. Channel activity (NPo), defined as the product of number of channel (N) and their open probability (Po), in outside-out patches also ran down in the absence of cytosolic ATP, and this run-down was significantly inhibited by cytosolic ATP (2mM), but not by ADP. These results provide evidence for a novel reguiation of rENaC activity, likely through the nonhydrolytic binding of cytosolic ATP.