1. 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

2. 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

3. 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

4. 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

5. 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

6. Background Many different types of PKC exist (  Chronic exposure to ethanol increases expression of PKC  and PKC . 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

7. The Question Which isozyme of PKC mediates the ethanol-induced up- regulation of L-type calcium channels?

8. The Tools PC12 cells transfected to express a peptide inhibitor of either PKC  or PKC  These are called V  and V , and exist in multiple versions (V  V  2, V  V  etc.) Experimental strategies include exposing cells chronically to ethanol at various concentrations and measuring calcium uptake

9. The Data

10. The V  fragment inhibits calcium uptake while the V  does not

11. The V  fragment inhibits L-type calcium channel up-regulation while the V  does not

12. The Conclusions Chronically exposing PC12 cells to ethanol: activates PKC  and PKC  Restores the “normal” calcium influx seen in the absence of ethanol Increases the number of cell-surface L-type channels Blocking the activity of PKC  but not PKC  prevents the effect of ethanol

13. The Conclusions (continued) These results identify PKC  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, PKC  may play a key role in controlling alcohol dependence and consumption

14. Next Step Can inhibitors of PKC  reduce or prevent ethanol consumption and the development of alcohol dependence in animals?