Specific and nonspecific effects of protein kinase C on the epithelial Na+channel

The Xenopus oocyte expression system was used to explore the mechanisms of inhibition of the cloned rat epithelial Nai channel (rENaC) by PKC (Awayda, M.S., I.I. Ismailov, B.K. Berdiev, C.M. Fuller, and D.J. Benos. 1996. J. G en. Physiol. 108:49-65) and to determine whether human ENaC exhibits simila r regulation. Effects of PKC activation on membrane and/or channel traffick ing were determined using impedance analysis as an indirect measure of memb rane area. hENaC-expressing oocytes exhibited an appreciable activation by hyperpolarizing voltages. This activation could be fit with a single expone ntial, described by a time constant (tau) and a magnitude (Delta I-v,). A s imilar but smaller magnitude of activation was also observed in oocytes exp ressing rENaC. This activation likely corresponds to the previously describ ed effect of hyperpolarizing voltage on gating of the native Na+ channel (P almer, L.G., and G. Frindt. 1996. J. Gen. Physiol. 107:35-45). Stimulation of PKC with 100 nM PMA decreased ill, in hENaC-expressing oocytes to a plat eau at 57.1 +/- 4.9% (n = 6) of baseline values at 20 min. Similar effects were observed in rENaCexpressing oocytes. PMA decreased the amiloride-sensi tive hENaC slope conductance (g(Na)) to 21.7 +/- 7.2% (n = 6) of baseline v alues at 30 min. This decrease was similar to that previously reported for rENaC. This decrease of g(Na) was attributed to a decrease of membrane capa citance (C-m), as well as the specific conductance (g(m)/C-m). The effects on g(m)/C-m reached a plateau within 15 min, at similar to 60% of baseline values. This decrease is likely due to the specific ability of PKC to inhib it ENaC. On the other hand, the decrease of C-m was unrelated to ENaC and i s likely an effect of PKC on membrane trafficking, as it was observed in EN aC-expressing as well as control oocytes. At lower PMA concentrations (0.5 nM), smaller changes of C-m were observed in rENaC- and hEhNaC-expressing o ocytes, and were preceded by larger changes of g(m) and by changes of g(m)/ C-m, indicating specific effects on ENaC. These findings indicate that PKC exhibits multiple and specific effects on ENaC, as well as nonspecific effe cts on membrane trafficking. Moreover, these findings provide the electroph ysiological logical basis for assessing channel-specific effects of PKC in the Xenopus oocyte expression system.