Protein kinase C-alpha and -epsilon down-regulate cell surface sodium channels via differential mechanisms in adrenal chromaffin cells

In cultured bovine adrenal chromaffin cells, our [H-3]saxitoxin ([H-3]STX) binding, immunoblot, and northern blot analyses specified protein kinase C (PKC) isoform-specific posttranscriptional and posttranslational mechanisms that direct down-regulation of cell surface Na channels. Immunoblot analys is showed that among 11 PKC isoforms, adrenal chromaffin cells contained on ly conventional (c)PKC-alpha, novel (n)PKC-epsilon, and atypical (a)PKC-xi. Treatment of adrenal chromaffin cells with 100 nM 12-O-tetradecanoylphorbo l 13-acetate (TPA) or 100 nM phorbol 12,13-dibutyrate (PDBu) caused a rapid (<15 min) and sustained (>15 h) translocation of PKC-alpha and -epsilon (b ut not -xi from cytosol to membranes, whereas a biologically inactive 4 alp ha-TPA had no effect. Thymeleatoxin (TMX), an activator of cPKC, produced s imilar membrane association of only PKC-alpha at 100 nM, with the potency o f TMX being comparable with those of TPA and PDBu. Treatment with either 10 0 nM TPA or 100 nM TMX reduced cell surface [H-3]STX binding to a comparabl e extent at 3, 6, and 12 h, whereas TPA lowered the binding to a greater ex tent than TMX at 15, 18, and 24 h; at 15 h, Go6976, a specific inhibitor of cPKC, completely blocked TMX-induced decrease of [H-3]STX binding while pr eventing by merely 57% TPA-induced decrease of [H-3]STX binding. Treatment with 100 nM TPA lowered the Na channel a-subunit mRNA level between 3 and 1 2 h, with its maximum 52% fall at 6 h, and it was accompanied by a subseque nt 61% rise of the beta(1)-subunit mRNA level at 24 h. Go6976 failed to pre vent TPA-induced reduction of the alpha-subunit mRNA level; TMX did not cha nge the alpha- and beta(1)-subunit mRNA levels throughout the 24-h treatmen t. Brefeldin A, an inhibitor of vesicular exit from the trans-Golgi network , augmented TPA- and TMX-induced decrease of [H-3]STX binding at 1 and 3 h. Our previous and present studies suggest that PKC down-regulates cell surf ace Na channels without altering the allosteric gating of Na channels via P KC isoform-specific mechanisms; cPKC-alpha promotes Na channel internalizat ion, whereas nPKC-epsilon decreases the alpha-subunit mRNA level by shorten ing the half-life of alpha-subunit mRNA without changing its gene transcrip tion.