Journal
JOURNAL OF NEUROCHEMISTRY
Volume 74, Issue 4, Pages 1674-1684Publisher
LIPPINCOTT WILLIAMS & WILKINS
DOI: 10.1046/j.1471-4159.2000.0741674.x
Keywords
sodium channel; protein kinase C-alpha and -epsilon; regulation; [H-3]saxitoxin binding; immunoblot; Northern blot
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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 analysis showed that among 11 PKC isoforms, adrenal chromaffin cells contained only conventional (c)PKC-alpha, novel (n)PKC-epsilon, and atypical (a)PKC-xi. Treatment of adrenal chromaffin cells with 100 nM 12-O-tetradecanoylphorbol 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 (but not -xi from cytosol to membranes, whereas a biologically inactive 4 alpha-TPA had no effect. Thymeleatoxin (TMX), an activator of cPKC, produced similar membrane association of only PKC-alpha at 100 nM, with the potency of TMX being comparable with those of TPA and PDBu. Treatment with either 100 nM TPA or 100 nM TMX reduced cell surface [H-3]STX binding to a comparable extent at 3, 6, and 12 h, whereas TPA lowered the binding to a greater extent 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 preventing 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 12 h, with its maximum 52% fall at 6 h, and it was accompanied by a subsequent 61% rise of the beta(1)-subunit mRNA level at 24 h. Go6976 failed to prevent TPA-induced reduction of the alpha-subunit mRNA level; TMX did not change the alpha- and beta(1)-subunit mRNA levels throughout the 24-h treatment. 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 surface Na channels without altering the allosteric gating of Na channels via PKC isoform-specific mechanisms; cPKC-alpha promotes Na channel internalization, whereas nPKC-epsilon decreases the alpha-subunit mRNA level by shortening the half-life of alpha-subunit mRNA without changing its gene transcription.
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