| Budget Amount *help |
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 1999: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1998: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Research Abstract |
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.
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