Voltage-gated Na+ channels set the threshold for action potential generation and are therefore good candidates to mediate forms of plasticity that affect the entire neuronal output. Although early studies led to the idea that Na+ channels were not subject to modulation, we now know that Na+ channel function is affected by phosphorylation. Furthermore, Na+ channel modulation is implicated in the control of input-output relationships in several types of neuron and seems to be involved in phenomena as varied as cocaine withdrawal, hyperalgesia and light adaptation. Here we review the available evidence for the regulation of Na+ channels by phosphorylation, its molecular mechanism, and the possible ways in which it affects neuronal function.

Voltage-gated Na+ channels are major targets of G protein-coupled receptor (GPCR)-initiated signaling cascades. These cascades act principally through protein kinase-mediated phosphorylation of the channel alpha subunit. Phosphorylation reduces Na+ channel availability in most instances without producing major alterations of fast channel gating. The nature of this change in availability is poorly understood. The results described here show that both GPCR- and protein kinase-dependent reductions in Na+ channel availability are mediated by a slow, voltage-dependent process with striking similarity to slow inactivation, an intrinsic gating mechanism of Na+ channels. This process is strictly associated with neuronal activity and develops over seconds, endowing neurons with a novel form of cellular plasticity shaping synaptic integration, dendritic electrogenesis, and repetitive discharge.

Phosphorylation of brain Na ϩ channel ␣ subunits by cAMPdependent protein kinase (PKA) decreases peak Na ϩ current in cultured brain neurons and in mammalian cells and Xenopus oocytes expressing cloned brain Na ϩ channels. We have studied PKA regulation of Na ϩ channel function by activation of D1-like dopamine receptors in acutely isolated hippocampal neurons using whole-cell voltage-clamp recording techniques. The D1 agonist SKF 81297 reversibly reduced peak Na ϩ current in a concentration-dependent manner. No changes in the voltage dependence or kinetics of activation or inactivation were observed. This effect was mediated by PKA, as it was mimicked by application of the PKA activator Sp-5,6-dichloro-1-␤-D-ribofuranosylbenzimidazole-3Ј,5Ј-monophosphorothioate (cBIMPS) and was inhibited by the specific PKA inhibitor peptide PKAI [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24] . cBIMPS had similar effects on type IIA brain Na ϩ channel ␣ subunits expressed in tsA-201 cells, but no effect was observed on a mutant Na ϩ channel ␣ subunit in which serine residues in five PKA phosphorylation sites in the intracellular loop connecting domains I and II (L I-II ) had been replaced by alanine. A single mutation, S573A, similarly eliminated cBIMPS modulation. Thus, activation of D1-like dopamine receptors results in PKA-dependent phosphorylation of specific sites in L I-II of the Na ϩ channel ␣ subunit, causing a reduction in Na ϩ current. Such modulation is expected to exert a profound influence on overall neuronal excitability. Dopaminergic input to the hippocampus from the mesocorticolimbic system may exert this influence in vivo.

The voltage-gated sodium channel is the site of action of more than six classes of neurotoxins and drugs that alter its function by interaction with distinct, allosterically coupled receptor sites. Batrachotoxin (BTX) is a steroidal alkaloid that binds to neurotoxin receptor site 2 and causes persistent activation. BTX binding is inhibited allosterically by local anesthetics. We have investigated the interaction of BTX with amino acid residues I1760, F1764, and Y1771, which form part of local anesthetic receptor site in transmembrane segment IVS6 of type IIA sodium channels. Alanine substitution for F1764 (mutant F1764A) reduces tritiated BTX-A-20-␣-benzoate binding affinity, causing a 60-fold increase in K d . Alanine substitution for I1760, which is adjacent to F1764 in the predicted IVS6 transmembrane alpha helix, causes only a 4-fold increase in K d . In contrast, mutant Y1771A shows no change in BTX binding affinity. For wildtype and mutant Y1771A, BTX shifted the voltage for halfmaximal activation Ϸ40 mV in the hyperpolarizing direction and increased the percentage of noninactivating sodium current to Ϸ60%. In contrast, these BTX effects were eliminated completely for the F1764A mutant and were reduced substantially for mutant I1760A. Our data suggest that the BTX receptor site shares overlapping but nonidentical molecular determinants with the local anesthetic receptor site in transmembrane segment IVS6 as well as having unique molecular determinants in transmembrane segment IS6, as demonstrated in previous work. Evidently, BTX conforms to a domain-interface allosteric model of ligand binding and action, as previously proposed for calcium agonist and antagonist drugs acting on L-type calcium channels.Voltage-gated sodium channels are responsible for the rapid depolarization during the initial phase of the action potential in most excitable cells (1). The brain sodium channel consists of a pore-forming ␣ subunit of Ϸ260 kDa associated with a 36-kDa ␤1 subunit and a 33-kDa ␤2 subunit (reviewed in ref.2). The primary structure of the ␣ subunit is organized in four homologous domains (I-IV) that contain six transmembrane segments (S1-S6) and an additional membrane-associated segment between S5 and S6 and are connected by large intracellular loops (3, 4).Potent neurotoxins specifically bind to more than six distinct but allosterically coupled receptor sites on the voltage-gated sodium channel and modify its function with lethal consequences (2, 5-7). The sodium channel is also the site of action for local anesthetics and certain anticonvulsant and antiarrhythmic drugs that are used therapeutically to decrease sodium channel activity in syndromes of hyperexcitability, such as cardiac arrhythmia and epileptic seizure (1,8,9). Although these sodium channel binding sites are distinct, allosteric interactions among the sites modulate drug and toxin binding (10-15). Neurotoxin receptor site 2 binds neurotoxins that cause persistent activation of sodium channels, including batrachotoxin (BTX), veratridine, ac...

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.