Phosphorylation at a Single Site in the Rat Brain Sodium Channel Is Necessary and Sufficient for Current Reduction by Protein Kinase A

Abstract

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...

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NaN, a novel voltage-gated Na channel, is expressed preferentially in peripheral sensory neurons and down-regulated after axotomy

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Developing a Neuronal Model for the Pathophysiology of Schizophrenia Based on the Nature of Electrophysiological Actions of Dopamine in the Prefrontal Cortex

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