G-protein-coupled regulation of a cloned epithelial Na⁺-channel expressed in xenopus oocytes

We have examined G-protein-related regulation of a cloned epithelial Na⁺ channel (αβy rENaC). Whole-cell currents were measured in oocytes impaled with a third injecting pipette, which assured reliable delivery of drugs. At a holding potential of -100 mV, injection of GTPvS (final concentration 50-100 /jM) caused an inhibition of the amiloride-sensitive ENaC current to 45.6 ±3.2 % of control (n = 5). This inhibition reached a plateau 5-10 min after GTPyS injection. GDPS, which lacks the gamma phosphate group, was without effect on ENaC current at 10 min post injection. Coinjection of GTPyS and bisindolylmaleimide (final concentration 2.55 //M), a high affinity PKC inhibitor (K; -10 nM), eliminated the inhibitory effect of GTPvS. Stimulation of PKC by bath application of 10-100 nM of phorbol-12-myristate-13-acetate (PMA), resulted in inhibition of amiloride-sensitive ENaC current. These results support the idea that broad stimulation of guanylate-binding proteins by GTPyS results in an inhibition of Na⁺ channel function by the dominant action of a PKC linked G-protein.