Voltage-gated sodium channels, which are responsible for the generation of action potentials in the brain, are phosphorylated by protein kinase C (PKC) in purified form. Activation of PKC decreases peak sodium current up to 80 percent and slows its inactivation for sodium channels in rat brain neurons and for rat brain type IIA sodium channel alpha subunits heterologously expressed in Chinese hamster ovary cells. These effects are specific for PKC because they can be blocked by specific peptide inhibitors of PKC and can be reproduced by direct application of PKC to the cytoplasmic surface of sodium channels in excised inside-out membrane patches. Modulation of brain sodium channels by PKC is likely to have important effects on signal transduction and synaptic transmission in the central nervous system.

The function of voltage-gated sodium channels that are responsible for action potential generation in mammalian brain neurons is modulated by phosphorylation by adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase (cA-PK) and by protein kinase C (PKC). Reduction of peak sodium currents by cA-PK in intact cells required concurrent activation of PKC and was prevented by blocking phosphorylation of serine 1506, a site in the inactivation gate of the channel that is phosphorylated by PKC but not by cA-PK. Replacement of serine 1506 with negatively charged amino acids mimicked the effect of phosphorylation. Conversion of the consensus sequence surrounding serine 1506 to one more favorable for cA-PK enhanced modulation of sodium currents by cA-PK. Convergent modulation of sodium channels required phosphorylation of serine 1506 by PKC accompanied by phosphorylation of additional sites by cA-PK. This regulatory mechanism may serve to integrate neuronal signals mediated through these parallel signaling pathways.

Voltage-gated sodium channels are responsible for generation of action potentials in excitable cells. Activation of protein kinase C slows inactivation of sodium channels and reduces peak sodium currents. Phosphorylation of a single residue, serine 1506, that is located in the conserved intracellular loop between domains III and IV and is involved in inactivation of the sodium channel, is required for both modulatory effects. Mutant sodium channels lacking this phosphorylation site have normal functional properties in unstimulated cells but do not respond to activation of protein kinase C. Phosphorylation of this conserved site in sodium channel alpha subunits may regulate electrical activity in a wide range of excitable cells.

SUMMARY1. Neurones were isolated from the hippocampus of adult guinea-pigs by enzymatic and mechanical treatment. The electrophysiological properties of these cells were examined immediately after dissociation by intracellular recordings using lowresistance electrodes (2-5 MC). Co2" consisted of a shift to the positive direction of the voltage dependence of the current.7. The delayed currents can be divided into Ca2+-dependent and Ca2+-independent components. The component persistent in the Co2+ solution (K-current) decayed slowly with maintained depolarization (time constant > 3 s). The current elicited by a fixed depolarization increased gradually as the hyperpolarizing pre-pulse increased in amplitude and reached its half-maximum value as the hyperpolarizing pre-pulse was at -87 mV.8. Ca2"-dependent outward currents can be divided into two types based on their voltage-dependent properties. One type had a high threshold (-30 mV) and did not decline during sustained depolarization. A second component with a lower threshold (-60 mV) decayed completely at holding potentials depolarized to -55 mV. Halfmaximal current was elicited by depolarization when the preceding hyperpolarization was at -95 mV. These Ca2_-dependent currents are named K1(Ca)-and K2(Ca)-currents.9. Outward currents in the hippocampal pyramidal cells can be separated into four types by their kinetic and pharmacological properties. These include the Acurrent, K-current, Kl(ca)-current and K2(ca)-current. All outward currents, except Kl(ca)-current, exhibit inactivating properties with the A-current and K2(ca)-current showing complete inactivation near the resting potential (about -55 mV). These results suggest that the resting levels of the hippocampal cells can significantly affect the repolarization process and thereby determine the firing pattern of the cells.

Voltage-gated ion channels regulate many physiological functions and are targets for a number of drugs. Patch-clamp electrophysiology is the standard method for measuring channel activity because it fulfils the requirements for voltage control, repetitive stimulation and high temporal resolution, but it is laborious and costly. Here we report an electro-optical technology and automated instrument, called the electrical stimulation voltage ion probe reader (E-VIPR), that measures the activity of voltage-gated ion channels using extracellular electrical field stimulation and voltage-sensitive fluorescent probes. We demonstrate that E-VIPR can sensitively detect drug potency and mechanism of block on the neuronal human type III voltage-gated sodium channel expressed in human embryonic kidney cells. Results are compared with voltage-clamp and show that E-VIPR provides sensitive and information-rich compound blocking activity. Furthermore, we screened approximately 400 drugs and observed sodium channel-blocking activity for approximately 25% of them, including the antidepressants sertraline (Zoloft) and paroxetine (Paxil).