Effect of protein kinase A-induced phosphorylation on the gating mechanismof the brain Na+ channel: Model fitting to whole-cell current traces

The activity of the voltage-gated Na+ channel is subjected to modulation th rough covalent modifications. It has been previously shown that brain Na+ c urrents are reduced following the activation of the protein kinase A (PKA) pathway, but the effect of the phosphorylation on the gating mechanism of t he 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, wh ich stimulates the PKA pathway. A minimal molecular model of the gating mec hanism of the Na+ channel is defined to fit the experimental data: it consi sts of three closed states, one opens state, and two inactivated states. We experimentally demonstrate that the kinetics of inactivation from the clos ed states are not affected by phosphorylation. The results obtained by comp uter fitting indicate that, among all the kinetic parameters describing the transitions between states, only one parameter is significantly modified i n the presence of forskolin, and corresponds to the acceleration of the ina ctivation from the open state. This conclusion is supported by the analysis of current traces obtained from cells in the presence of a phosphatase inh ibitor or loaded with the PKA catalytic unit, and is in agreement with prev iously reported single channel records.