Phosphorylation of brain Na+ channels by protein kinase C (PKC) decreases peak Na+ current and slows macroscopic inactivation, but receptor-activated modulation of Na+ currents via the PKC pathway has not been demonstrated. We have examined modulation of Na+ channels by activation of muscarinic receptors in acutely-isolated hippocampal neurons using whole-cell voltage-clamp recording. Application of the muscarinic agonist carbachol reduced peak Na+ current and slowed macroscopic inactivation at all potentials, without changing the voltage-dependent properties of the channel. These effects were mediated by PKC, since they were eliminated when the specific PKC inhibitor (PKCI19-36) was included in the pipette solution and mimicked by the extracellular application of the PKC activator, OAG. Thus, activation of endogenous muscarinic receptors on hippocampal neurons strongly modulates Na+ channel activity by activation of PKC. Cholinergic input from basal forebrain neurons may have this effect in the hippocampus in vivo.

Persistent Na+ currents are thought to be important for integration of neuronal responses. Here, we show that betagamma subunits of G proteins can induce persistent Na+ currents. Coexpression of G beta2gamma3, G beta1gamma3, or G beta5gamma3, but not G beta1gamma1 subunits with rat brain type IIA Na+ channel alpha subunits in tsA-201 cells greatly enhances a component of Na+ current with a normal voltage dependence of activation but with dramatically slowed and incomplete inactivation and with steady-state inactivation shifted +37 mV. Synthetic peptides containing the proposed G betagamma-binding motif, Gln-X-X-Glu-Arg, from either adenylyl cyclase 2 or the Na+ channel alpha subunit C-terminal domain reversed the effect of G beta2gamma3 subunits. These results are consistent with direct binding of G betagamma subunits to the C-terminal domain of the Na+ channel, stabilizing a gating mode responsible for slowed and persistent Na+ current. Modulation of Na+ channel gating by G betagamma subunits is expected to have profound effects on neuronal excitability.

Na+ channels in acutely dissociated rat hippocampal neurons and in Chinese hamster ovary (CHO) cells transfected with a cDNA encoding the a subunit of rat brain tpe IIA Na+ channel [14][15][16]. In heart, the opening of an inwardly rectifying K+ channel by muscarinic acetylcholine receptors requires GTP and a pertussis toxin (PTX)-sensitive G protein (17)(18)(19)(20), and 3-adrenergic receptors modulate Na+ channels by parallel pathways involving cAMP-dependent protein phosphorylation and possibly direct interaction with G, (21,22 EXPERIMENTAL PROCEDURES Cell Culture. Hippocampal neurons were acutely dissociated from 7-to 21-day-old Sprague-Dawley rats as described by Kuo and Bean (24). CNaIIA-1 cells were maintained as described (6).Electrophysiological Recording. Whole-cell voltage-clamp recordings (25) were obtained as described (6) using micropipettes with resistances of 1-2 Mfl in our internal solutions. Capacity transients were canceled and series resistance was compensated (>80%6) using the internal voltage-clamp circuitry. Remaining linear capacity transients as well as leakage currents were subtracted by the P/4 procedure. Conductance-voltage (g-V) relationships were calculated from peak current vs. voltage (I-V) relationships according to g = I/(V -Vr), where I is the peak current measured at voltage V, and Vr is the measured reversal potential. Normalized conductance-voltage relationships and inactivation curves were fit with a Boltzmann relation, 1/(1 + exp[(V -Vl/2)/k]), where

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