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Protein Kinase C Mediates Ethanol-Induced Up-regulation of L-Type Calcium Channels. Journal of Biological Chemistry Vol. 273 No. 26 pp 16409 – 16414 1998. Background. Chronic alcohol exposure produces tolerance to the effects of alcohol BAC above 100 mM leads to coma in non-alcoholic
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Protein Kinase C Mediates Ethanol-Induced Up-regulation of L-Type Calcium Channels Journal of Biological Chemistry Vol. 273 No. 26 pp 16409 – 16414 1998
Background • Chronic alcohol exposure produces tolerance to the effects of alcohol • BAC above 100 mM leads to coma in non-alcoholic • Alcoholics can seem normal or only slightly intoxicated at BAC of 100 – 150 mM BAC blood alcohol content
Background • Voltage-dependent calcium channels are membrane proteins that open to allow influx of calcium ions when a neuron depolarizes • There are several types of VDCC, distinguished by their response to depolarization level, location, and sensitivity to various compounds: • L type: focus of this article, found in neurons • T type • N type • R • P/Q
Background • Acute exposure of neuronal cell cultures to ethanol inhibits calcium influx through L-type channels • Chronic exposure leads to tolerance – a recovery of the “normal” calcium influx • Due to increased number of L-type calcium channels on cell surface
Background • Protein kinase C is a serine/threonine kinase involved in many signaling pathways related to: • Cell growth & differentiation • Neurotransmitter release • Receptor regulation • Ion channel modulation • Gene expression
Background • Many different types of PKC exist (a, bi, b2, g, d, e, z, l, m) • Chronic exposure to ethanol increases expression of PKCd and PKCe. • Compounds that inhibit activity of all PKCs can prevent the effect of ethanol on L-type calcium channels • Thus, the effect of ethanol on calcium channels may be mediated through its effect on PKC
The Question • Which isozyme of PKC mediates the ethanol-induced up-regulation of L-type calcium channels?
The Tools • PC12 cells transfected to express a peptide inhibitor of either PKCd or PKCe • These are called Vd and Ve, and exist in multiple versions (Vd1, Vd2, Ve1, Ve2, etc.) • Experimental strategies include exposing cells chronically to ethanol at various concentrations and measuring calcium uptake
The Vd fragment inhibits calcium uptake while the Ve does not
The Vd fragment inhibits L-type calcium channel up-regulation while the Ve does not
The Conclusions • Chronically exposing PC12 cells to ethanol: • activates PKCd and PKCe • Restores the “normal” calcium influx seen in the absence of ethanol • Increases the number of cell-surface L-type channels • Blocking the activity of PKCd but not PKCeprevents the effect of ethanol
The Conclusions (continued) • These results identify PKCd as a regulator of L-type calcium channel density and a mediator of cellular adaptation to ethanol. • Since L-type calcium channels modulate drinking behavior and contribute to withdrawal, PKCd may play a key role in controlling alcohol dependence and consumption
Next Step • Can inhibitors of PKCd reduce or prevent ethanol consumption and the development of alcohol dependence in animals?