The mouse Scn8a sodium channel and its ortholog Na6 in the rat are abundantly expressed in the CNS. Mutations in mouse Scn8a result in neurological disorders, including paralysis, ataxia, and dystonia. In addition, Scn8a has been observed to mediate unique persistent and resurgent currents in cerebellar Purkinje cells (Raman et al., 1997). To examine the functional characteristics of this channel, we constructed a full-length cDNA clone encoding the mouse Scn8a sodium channel and expressed it in Xenopus oocytes. The electrophysiological properties of the Scn8a channels were compared with those of the Rat1 and Rat2 sodium channels. Scn8a channels were sensitive to tetrodotoxin at a level comparable to that of Rat1 or Rat2. Scn8a channels inactivated more rapidly and showed differences in their voltage-dependent properties compared with Rat1 and Rat2 when only the alpha subunits were expressed. Coexpression of the beta1 and beta2 subunits modulated the properties of Scn8a channels, but to a lesser extent than for the Rat1 or Rat2 channels. Therefore, all three channels showed similar voltage dependence and inactivation kinetics in the presence of the beta subunits. Scn8a channels coexpressed with the beta subunits exhibited a persistent current that became larger with increasing depolarization, which was not observed for either Rat1 or Rat2 channels. The unique persistent current observed for Scn8a channels is consistent with the hypothesis that this channel is responsible for distinct sodium conductances underlying repetitive firing of action potentials in Purkinje neurons.

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.

Voltage-gated sodium channels in the mammalian CNS initiate and propagate action potentials when excitatory inputs achieve threshold membrane depolarization. There are multiple sodium channel isoforms expressed in rat brain (types I, II, III, 6, and NaG). We have constructed a full-length cDNA clone encoding type I and compared the electrophysiological properties of type I (Rat1) and II (Rat2) channels in the absence and presence of the two accessory subunits beta1 and beta2. Injection into Xenopus oocytes of RNA encoding Rat1 resulted in functional sodium currents that were blocked by tetrodotoxin, with Kapp = 9.6 nM. Rat1 sodium channels had a slower time course of fast inactivation than Rat2. Coexpression of beta1 accelerated inactivation of both Rat1 and Rat2, resulting in comparable inactivation kinetics. Rat1 recovered from fast inactivation more rapidly than Rat2, regardless of whether beta1 or beta2 was present. The voltage dependence of activation was similar for Rat1 and Rat2 without the beta subunits, but it was more positive for Rat1 when beta1 and beta2 were coexpressed. The voltage dependence of inactivation was more positive for Rat1 than for Rat2, and coexpression with beta1 and beta2 accentuated that difference. Finally, sodium current amplitudes were reduced by 7-9% for both Rat1 and Rat2 channels when protein kinase A phosphorylation was induced. It has been suggested previously that Rat1 and Rat6 channels mediate transient and maintained sodium conductances, respectively, in Purkinje cells, and the electrophysiological properties of Rat1 currents are consistent with a role for this channel in mediating the rapidly inactivating, transient current.

Voltage-gated sodium channels respond to excitatory inputs in nerve cells, generating spikes of depolarization at axon hillock regions and propagating the initial rising phase of action potentials through axons. It previously has been shown that protein kinase A (PKA) attenuates sodium current amplitude 20-50% by phosphorylating serines located in the I-II linker of the sodium channel. We have tested the individual contributions of five PKA consensus sites in the I-II linker by measuring sodium currents expressed in Xenopus oocytes during conditions of PKA induction. PKA was induced by perfusing oocytes with a cocktail that contained forskolin, chlorophenylthio-cAMP, dibutyryl-cAMP, and 3-isobutyl-1-methylxanthine. Phosphorylation at the second PKA site (serine-573) was necessary and sufficient to diminish sodium current amplitude. Phosphorylation at the third and fourth positions (serine-610 and serine-623) reduced current amplitude, but the effect was considerably smaller at those positions. Introduction of a negative charge at site 2 by substitution of serine-573 with an aspartate constitutively reduced the basal level of sodium current, indicating that the attenuation of sodium current by phosphorylation of site 2 by PKA results from the introduction of a negative charge at this site. Key words: ion channel; modulation; cAMP; site-directed mutagenesis; sodium channel; phosphorylation; protein kinase A; Xenopus oocytes; forskolinVoltage-gated sodium channels play a key role in the transmission of signals through electrically excitable cells. If the spatial summation of excitatory and inhibitory inputs exceeds a threshold level of membrane depolarization, sodium channels that are localized at high density in the axon hillock region of nerve cells are activated, resulting in initiation of an action potential. Therefore, one mechanism by which electrical excitability can be regulated is by altering the activity of sodium channels.Phosphorylation by protein kinase A (PK A) is a common regulatory mechanism that is observed for a large number of proteins. In the case of the voltage-gated rat brain sodium channel, PK A phosphorylation modifies channel f unction by reducing the peak current amplitude but without affecting sodium current kinetics or the voltage dependence of conductance or inactivation (Gershon et al., 1992;Li et al., 1992;Schiffmann et al., 1995;Smith and Goldin, 1996). When purified PK A was applied to patches that were excised from transfected Chinese hamster ovary (CHO) cells, sodium current magnitude was reduced by 40 -50%, resulting from a decrease in the open probability of the channels (Li et al., 1992). Similarly, when the sodium channel was expressed in Xenopus oocytes, PK A induction either by forskolin or by isoproterenol stimulation of a coexpressed ␤ 2 -adrenergic receptor reduced current amplitude by 20 -30% (Gershon et al., 1992;Smith and Goldin, 1996). All other electrophysiological properties of the channel were unchanged. Finally, when PKA was induced in cultured striatal neurons fr...

Excitability has long been recognized as the basis for rapid signaling in the mature nervous system, but roles of channels and receptors in controlling slower processes of differentiation have been identified only more recently. Voltage-dependent and transmitter-activated channels are often expressed at early stages of development prior to synaptogenesis, and allow influx of Ca(2+). Here we examine the functions of spontaneous transient elevations of intracellular Ca(2+) in embryonic neurons. These Ca(2+) transients abruptly raise levels of Ca(2+) as much as tenfold, for brief periods, repeatedly, and can be highly localized. Like cloudbursts on the developing landscape, Ca(2+) transients modulate growth and stimulate differentiation, in a frequency-dependent manner, probably by changes in phosphorylation or proteolysis of regulatory and structural proteins in local regions. We review the mechanisms by which Ca(2+) transients are generated and their effects in regulating motility via the cytoskeleton and differentiation via transcription.

The epithelial sodium channel (ENaC), a heterotrimeric complex composed of ␣, ␤, and ␥ subunits, belongs to the ENaC/ degenerin family of ion channels and forms the principal route for apical Na ؉ entry in many reabsorbing epithelia. Although high affinity ENaC blockers, including amiloride and derivatives, have been described, potent and specific small molecule ENaC activators have not been reported. Here we describe compound S3969 that fully and reversibly activates human ENaC (hENaC) in an amiloride-sensitive and dose-dependent manner in heterologous cells. Mechanistically, S3969 increases hENaC open probability through interactions requiring the extracellular domain of the ␤ subunit. hENaC activation by S3969 did not require cleavage by the furin protease, indicating that nonproteolyzed channels can be opened. Function of ␣␤G37S␥ hENaC, a channel defective in gating that leads to the salt-wasting disease pseudohypoaldosteronism type I, was rescued by S3969. Small molecule activation of hENaC may find application in alleviating human disease, including pseudohypoaldosteronism type I, hypotension, and neonatal respiratory distress syndrome, when improved Na ؉ flux across epithelial membranes is clinically desirable.

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The voltage-sensitive rat brain sodium channel is known to be phosphorylated by adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase A (PKA), but the functional significance of that phosphorylation is unknown. We have shown that rat brain sodium channel currents expressed in Xenopus oocytes were enhanced by induction of PKA activity. Stimulation of the beta 2-adrenergic receptor or treatment with dibutyryl cAMP resulted in increased sodium current amplitudes without affecting the voltage dependence of channel activation or inactivation. These increases were completely blocked by preinjection of protein kinase inhibitor, a specific inhibitor of PKA. Injection of phosphatase into the oocytes resulted in a significant decrease in sodium current amplitude, indicating that phosphorylation is important for basal levels of sodium channel activity in oocytes. The enhancement was specific for the rat brain IIA sodium channel, because currents expressed from the rat muscle microI sodium channel were not enhanced by the same procedures. These data demonstrate a modulatory role of PKA phosphorylation on brain sodium channel function and suggest a means by which the electrical excitability of cells may be regulated.