| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2001: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 2000: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
I have previously found that cytosolic ATP regulates activity of the rat epithelial Na1 channel (αβγ-rENaC) heterologously expressed in NIH-3T3 cells. Using both patch-clamp and biochemical techniques, I have now shown that the ATP regulation of rENaC is also seen in rENaC stably expressed.in MDCK epithelial cells and that the regulation is likely mediated through a non-hydrolytic nucleotide binding to α-rENaC. In order to identify the ATP binding sites I have generated cell lines expressing mutant α-rENaCs.
ENaC is known to be regulated by cystic fibrosis transmembrane conductance regulator (CFTR), which functions as a cAMP-regulated, ATP-dependent Cl^-channel Since CFTR is localized in the apical membrane of epithelial cells and also possesses ATPase activity necessary for Cr conductance, I hypothesized that ENaC regulation by CFTR may be also mediated by a mechanism through which cytosolic ATP regulates ENaC. To begin with, I first characterized electrophysiological properties of rENaC in NIH3T3 cells coexpressing rENaC and CFTR using patch-clamp techniques. Electrophysiological properties of rENaC such as ion selectivity, amiloride-sensitivity were similar to those in NIH3T3 cells expressing rENaC-alone. I also showed that CFTR activity was seen in NIH3T3 cells coexpressing rENaC and CFTR, but not in the cells expressing rENaC alone. In order to study functional interaction ofrENaC and CFTR in native epithelial cells, native rENaC currents in freshly isolated rat colonic surface epithelial cells, where rENaC was first cloned and where CFTR has been known to be expressed, were first functionally characterized using whole-cell patch-clamp techniques. Electrophysiological properties such as amiloride-sensitivity, ion selectivity of native amiloride-sensitive currents were strikingly similar to those of the cloned rENaC expressed in MDCK and NIH-3T3 cells.
By using these experimental systems, I am going to systematically test the hypothesis mentioned above.
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