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Original Article

^a Department of Medicine and Department of Physiology, Tulane University School of Medicine, New Orleans, Louisiana 70112
Department of Medicine, SL 35, Tulane University School of Medicine, 1430 Tulane Avenue, New Orleans, LA 70112.504-587-2188

mawayda{at}mailhost.tcs.tulane.edu

The Xenopus oocyte expression system was used to explore the mechanisms of inhibition of the cloned rat epithelial Na⁺ channel (rENaC) by PKC (Awayda, M.S., I.I. Ismailov, B.K. Berdiev, C.M. Fuller, and D.J. Benos. 1996. J. Gen. Physiol. 108:49–65) and to determine whether human ENaC exhibits similar regulation. Effects of PKC activation on membrane and/or channel trafficking were determined using impedance analysis as an indirect measure of membrane area. hENaC-expressing oocytes exhibited an appreciable activation by hyperpolarizing voltages. This activation could be fit with a single exponential, described by a time constant () and a magnitude (I _V). A similar but smaller magnitude of activation was also observed in oocytes expressing rENaC. This activation likely corresponds to the previously described effect of hyperpolarizing voltage on gating of the native Na⁺ channel (Palmer, L.G., and G. Frindt. 1996. J. Gen. Physiol. 107:35–45). Stimulation of PKC with 100 nM PMA decreased I_V in hENaC-expressing oocytes to a plateau at 57.1 ± 4.9% (n = 6) of baseline values at 20 min. Similar effects were observed in rENaC-expressing oocytes. PMA decreased the amiloride-sensitive hENaC slope conductance (g_Na) to 21.7 ± 7.2% (n = 6) of baseline values 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 capacitance (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 60% of baseline values. This decrease is likely due to the specific ability of PKC to inhibit ENaC. On the other hand, the decrease of C_m was unrelated to ENaC and is likely an effect of PKC on membrane trafficking, as it was observed in ENaC-expressing as well as control oocytes. At lower PMA concentrations (0.5 nM), smaller changes of C_m were observed in rENaC- and hENaC-expressing oocytes, 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 effects on membrane trafficking. Moreover, these findings provide the electrophysiological basis for assessing channel-specific effects of PKC in the Xenopus oocyte expression system.

Key Words: epithelial Na⁺ channel • Xenopus oocytes • protein kinase C • impedance analysis • trafficking

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