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

通过其核苷酸结合域调节大鼠上皮 NaD1+D1 通道

基本信息

批准号：
10670053
负责人：
ISHIKAWA Toru
金额：
$ 2.05万
依托单位：
HOKKAIDO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10670053/
关键词：
ENaC ATP Patch-clamp Patch―clamp

项目摘要

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.

上皮细胞Na + Na + D1通道（ENaC）由3个亚基（αβγ）组成，在多种Na + D1-Na + D1吸收上皮细胞中表达，在水盐平衡和血压调节中起重要作用。应用膜片钳技术，研究了细胞内ATP对NIH-3 T3细胞中稳定表达的大鼠αβγENaC（rENaC）活性的影响。在这些细胞中的rENaC的宏观和微观阿米洛利敏感电流的生物物理特性相似的异源表达在非洲爪蟾卵母细胞和MDCK细胞。在传统的全细胞膜片钳记录，透析的细胞与Cs-谷氨酸盐丰富的移液器溶液不含ATP引起的向下的向内的全细胞电流归因于rENaC的活动，下降约50%，在5分钟内。向下的抑制细胞内应用ATP（102 mM），抑制不依赖于存在的镁离子D12+镁离子D1的移液器溶液。不可水解的ATP类似物AMP-PNP和水解性差的类似物ATP-γS（2 mM）也能有效地防止电流下降，而GTP、UTP则不能。ADP或AMP（2 mM）相对无效，其有效性顺序为ATP > ADP = GTP > UTP =AMP。在单通道水平上，单一的rENaC通道电导在外向补丁不受细胞溶质ATP，镁离子D12+ β-D1浓度或取代与各种核苷酸。通道活性（NPo），定义为通道数（N）和它们的开放概率（Po）的乘积，在没有胞浆ATP的情况下，由外向外的斑块中也下降，并且这种下降被胞浆ATP（2 mM）显著抑制，但不被ADP抑制。这些结果为rENaC活性的新调节提供了证据，可能是通过胞浆ATP的非水解结合。