Functional Effects of Protein Kinase C Activation on the Human Cardiac Na sup + Channel

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Abstract
The cardiac Na sup + current plays an important role in determining normal and abnormal impulse propagation in the heart. We have investigated the effects of protein kinase C (PKC) activation on the recombinant human cardiac Na sup + channel (hH1) following heterologous expression in Xenopus laevis oocytes. Phorbol 12-myristate 13-acetate (PMA), which directly activates PKC, reduced current amplitude at all test potentials (43 +/- 12% at -10 mV). In contrast to the rat brain IIA (rBIIA) channel, there was no apparent change in either macroscopic Na sup + current decay or the voltage dependence of channel gating. Further experiments indicate that the effects of PMA were mediated by PKC activation: (1) an inactive stereoisomer, 4 alpha-PMA, had no effect; (2) preincubation with the protein kinase inhibitor chelerythrine prevented the PMA effects; and (3) a hydrolyzable diacylglycerol analogue, 1-oleoyl-2-acetyl-glycerol, also reduced current (22 +/- 5%). In addition, when the alpha1B-adrenergic receptor was coexpressed with hH1, the alpha-receptor agonist methoxamine reduced hH1 current (45 +/- 10%), an effect that could be eliminated by chelerythrine preincubation. When a conserved consensus PKC site (serine 1503) in the III-IV interdomain linker thought to be responsible for the PKC effects on rBIIA was mutated, PMA still reduced Na sup + current, but the magnitude of the effect was smaller compared with that for the wild-type channel. Similar findings were obtained with alpha1-receptor stimulation following receptor coexpression with the mutant channel. We conclude that activation of PKC modulates the human cardiac Na sup + channel by at least two mechanisms, one similar to that seen with rat brain channels, involving a conserved putative PKC site, and a second more specific to the cardiac isoform.