Effect of protein kinase A-induced phosphorylation on the gating mechanism of the brain Na+ channel: model fitting to whole-cell current traces.
The activity of the voltage-gated Na+ channel is subjected to modulation through covalent modifications. It has been previously shown that brain Na+ currents are reduced following the activation of the protein kinase A (PKA) pathway, but the effect of the phosphorylation on the gating mechanism of the channel has not been demonstrated so far. In this study, we analyze the whole-cell Na+ current recorded in the absence or presence of forskolin, which stimulates the PKA pathway. A minimal molecular model of the gating mechanism of the Na+ channel is defined to fit the experimental data: it consists of three closed states, one open state, and two inactivated states. We experimentally demonstrate that the kinetics of inactivation from the closed states are not affected by phosphorylation. The results obtained by computer fitting indicate that, among all the kinetic parameters describing the transitions between states, only one parameter is significantly modified in the presence of forskolin, and corresponds to the acceleration of the inactivation from the open state. This conclusion is supported by the analysis of current traces obtained from cells in the presence of a phosphatase inhibitor or loaded with the PKA catalytic unit, and is in agreement with previously reported single channel records.
d'Alcantara
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Institut de Recherche Interdisciplinaire en Biologie humaine et Nucléaire, Faculté de Médecine, Université libre de Bruxelles, Brussels, Belgium. pdalcant@ulb.ac.be
Schiffmann
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Swillens
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D000818:Animals; D001921:Brain / Q000378:metabolism*; D002478:Cells, Cultured; D005576:Colforsin / Q000031:analogs & derivatives / Q000494:pharmacology; D003198:Computer Simulation; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D051937:Dopamine and cAMP-Regulated Phosphoprotein 32; D004594:Electrophysiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015640:Ion Channel Gating / Q000187:drug effects*; D007700:Kinetics; D009419:Nerve Tissue Proteins / Q000378:metabolism; D018408:Patch-Clamp Techniques; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D051381:Rats; D017208:Rats, Wistar; D015222:Sodium Channels / Q000378:metabolism*
D051937:Dopamine and cAMP-Regulated Phosphoprotein 32; D004791:Enzyme Inhibitors; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; D015222:Sodium Channels; D005576:Colforsin; D017868:Cyclic AMP-Dependent Protein Kinases; D010749:Phosphoprotein Phosphatases; C047983:1,9-dideoxyforskolin