Luminal sodium entrythrough amiloride-sensitive, highly-selective sodium channels (ENaC) in the apical membrane of distal nephron epithelial cells is the rate-limiting step for hormonally

We studied cellular phosphatase inhibitors, okadaic acid (OKA), calyculin A, and microcystin, on ENaC in A6 renal cells. OKA increased amiloride-sensitive current after about 30 minutes with maximal stimulation at 1-2 hours. Fluctuation analysis of cell-attached patches containing a large number of ENaC yielded power spectra with corner frequencies in untreated cells almost twice as large as in cells pretreated for 30 minutes with OKA implying an increase in single channel open probability (P o) that doubled after okadaic acid. Single channel analysis showed that in cells pretreated with okadaic acid, P o and mean open time approximately doubled. Two other phosphatase inhibitors, calyculin A and microcystin, had similar effects on open probability and mean open time. An analog of okadaic acid, okadaone, which does not inhibit phosphatases had no effect. Pretreatment with 10 nM OKA, which blocks PP2A but not PP1 in mammalian cells, had no effect even though both phosphatases are present in A6 cells. Several proteins were differentially phosphorylated after OKA, but ENaC subunit phosphorylation did not increase. We conclude that, in A6 cells, there is an OKA-sensitive phosphatase which suppresses ENaC activity by altering the phosphorylation of a regulatory molecule associated with the channel.