INTRODUCTION

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Understanding biochemical mechanisms that underlie alcohol tolerance and dependence may lead to new treatments for alcoholism. In nonalcoholic persons, intoxication develops at blood alcohol levels of 10-35 mM, and acute tolerance develops rapidly, so that after a few hours, an individual can appear sober at alcohol levels that previously caused intoxication (1). The mechanism for acute tolerance may involve activation of Fyn kinase and reduced sensitivity of tyrosine-phosphorylated N-methyl-D-aspartate receptors to inhibition by alcohol (2). Chronic tolerance is characteristic of alcoholism, and its magnitude in alcoholics can be quite striking. For example, blood alcohol concentrations above 100 mM produce coma in a nonalcoholic person, whereas human alcoholics may appear sober or only mildly intoxicated with blood levels of 100-150 mM (3, 4).

The ability of ethanol to alter the function of neuronal voltage-dependent calcium channels appears to contribute to chronic tolerance. In several neural preparations, ethanol inhibits voltage-dependent calcium influx and calcium currents (5-13). Chronic exposure results in the development of tolerance to the inhibitory actions of ethanol on calcium channels (6, 7). The mechanisms underlying this adaptive response have been studied most in the neural cell line PC12. In PC12 cells, prolonged exposure to 25-200 mM ethanol for 2-6 days produces a reversible concentration- and time-dependent increase in K⁺-evoked ⁴⁵Ca²⁺ uptake (8, 9, 14) and L-type calcium currents (14) measured in the absence of ethanol. This is associated with a corresponding increase in the number of binding sites for dihydropyridine Ca²⁺ channel antagonists (8, 9), suggesting that cells adapt to chronic ethanol exposure by increasing expression of functional L-type calcium channels. Similar increases in dihydropyridine binding have been detected in ethanol-treated NG108-15 neuroblastoma × glioma cells (15) and in brain membranes from ethanol-treated rats (16).

Up-regulation of L-type channels could promote alcohol consumption since L-channel antagonists reduce consumption in animals (17-20). Increases in L-type calcium channels may also contribute to the intense neuronal hyperexcitability observed during alcohol withdrawal (21). A role for L-type channels in alcohol withdrawal syndromes is supported by evidence that L-channel antagonists reduce tremors, seizures, and mortality in alcohol-dependent rodents deprived of ethanol (22-24). Moreover, ethanol-induced increases in binding sites for L-channel antagonists are greater in mice bred for severe alcohol withdrawal seizures than in mice bred for minor signs of alcohol withdrawal (25). These findings suggest that L-channel up-regulation plays an important role in alcohol dependence.

Protein kinase C (PKC)¹ is a family of phospholipid-dependent serine/threonine kinases involved in cell growth and differentiation, neurotransmitter release and receptor regulation, ion channel modulation, and gene expression (26). Eleven PKC isozymes have been identified (, I, II, , , , , , , , and µ), and they differ in structure and requirements for activation by diacylglycerol and calcium (26-28). In PC12 cells, we found that up-regulation of L-type channels by ethanol is inhibited by the kinase inhibitors sphingosine and polymyxin B (29). The effect of sphingosine is reversed by phorbol esters that activate all PKC isozymes except PKC and PKC (26), suggesting that ethanol-induced up-regulation of L-type channels requires activation of a phorbol ester-sensitive PKC. We also found that chronic ethanol exposure increases total PKC activity, high affinity phorbol ester binding, and PKC-mediated phosphorylation in PC12 cells (30). This is associated with a selective increase in immunoreactivity (30) and mRNA (31) for two PKC isozymes, PKC and PKC. Taken together, these findings suggest that chronic exposure to ethanol up-regulates functional L-type channels through a mechanism that involves ethanol-induced increases in expression of PKC or PKC.

In this paper, we examined whether PKC or PKC mediates ethanol-induced increases in L-type channels by using stably transfected PC12 cell lines that express the V1 or V1 fragment, which are derived from the first variable domains of PKC and PKC, respectively. These fragments selectively inhibit phorbol ester-induced translocation of the corresponding isozyme, and the V1 fragment specifically prevents phorbol ester-mediated inhibition of contraction in cultured cardiac myocytes (32) and enhancement of nerve growth factor-induced neurite outgrowth and mitogen-activated protein kinase activation by ethanol or phorbol esters in PC12 cells (33). We found that chronic exposure to ethanol increased K⁺-stimulated ⁴⁵Ca²⁺ uptake and dihydropyridine binding in PC12 cells, vector-transfected cells, and cells expressing V1, but not in cells expressing V1. We also found that overexpression of PKC increased dihydropyridine binding, but did not enhance K⁺-stimulated ⁴⁵Ca²⁺ uptake. These results demonstrate that PKC mediates increases in L-type calcium channels induced by chronic exposure to ethanol. These results are the first to demonstrate a functional role for PKC in neural cells.

	EXPERIMENTAL PROCEDURES

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Cell Culture-- In these experiments, we used PC12 cells obtained from Dr. John A. Wagner (Cornell University, New York). Cells were grown at 37 °C in plastic tissue culture flasks in Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum, 10% horse serum, 50 units/ml penicillin, 50 µg/ml streptomycin, and 2 mM glutamine in a humidified atmosphere of 90% air and 10% CO₂. Cells were cultured with ethanol in tightly capped tissue culture flasks or in six-well plates wrapped in Parafilm, and the medium was changed daily as described previously (8, 29). Parallel control samples were cultured in a similar manner without ethanol. Stably transfected cell lines that overexpress PKC or PKC have been described previously (34). Cell lines that express Flag epitope-tagged V1 or V1 were created and analyzed by reverse transcriptase-polymerase chain reaction and Western analysis for the Flag epitope tag as described (33).

Phorbol Ester-stimulated Translocation of PKC-- Cells (4 × 10⁶) were plated on 100-mm² plastic tissue culture plates. After 2 days, the plates were rinsed with 10 ml of Dulbecco's modified Eagle's medium and incubated with or without 30 nM phorbol 12-myristate 13-acetate (PMA) for 2 min at 37 °C. Cells were rinsed twice with Ca²⁺- and Mg²⁺-free phosphate-buffered saline and scraped into ice-cold buffer containing 20 mM Tris-HCl, pH 7.5, 2 mM EDTA, 10 mM EGTA, 40 µg/ml leupeptin, 40 µg/ml aprotinin, 20 µg/ml soybean trypsin inhibitor, and 1 mM phenylmethylsulfonyl fluoride. Cells were homogenized in a Teflon-glass homogenizer, and sucrose was added to a final concentration of 250 mM. Samples were then centrifuged at 150,000 × g for 1 h, and the supernatant was saved as the cytosolic fraction. The pellet was dispersed by sonication, and samples of supernatant and pellet derived from 100 µg of crude homogenate were analyzed by Western analysis as described (33).

K⁺-stimulated ⁴⁵Ca²⁺ Uptake-- PC12 cells were plated onto poly-D-lysine-coated six-well tissue culture plates at a density of 0.8-1.6 × 10⁶ cells/well. After 24 h, cells were cultured for another 1-6 days in the presence or absence of ethanol. On the day of assay, cells were rinsed twice with 1 ml of 5 mM KCl buffer (85 mM NaCl, 5 mM KCl, 45 mM choline chloride, 2 mM CaCl₂, 5 mM glucose, and 25 mM HEPES, pH 7.4) at room temperature, and incubated in the same buffer for 25 min. Cells were then incubated at 25 °C in 5 or 50 mM KCl buffer containing 0.75 µCi of ⁴⁵Ca²⁺. The composition of the 50 mM KCl buffer was identical to that of the 5 mM KCl buffer except that choline chloride was replaced by KCl. After incubation for 2.5 min, cells were washed four times with 2.5 ml of ice-cold 5 mM KCl buffer and incubated overnight with 1 ml of 1 M NaOH. Radioactivity in neutralized samples was measured by liquid scintillation counting, and protein levels were determined by the method of Lowry et al. (35). K⁺-stimulated uptake is defined as the difference between uptake in 50 and 5 mM KCl buffers and represents 85 ± 1% (n = 10) of total uptake in 50 mM KCl buffer.

(+)-[³H]PN200-110 Binding-- Binding of (+)-[³H]PN200-110 to whole cells was measured as described previously (36). Specific binding was determined by subtracting binding in the presence of 1 µM nimodipine from binding in its absence and represents 51 ± 2% (n = 10) of total binding at 50 pM radioligand.

	RESULTS

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GF 109203X Prevents Ethanol-induced Increases in K⁺-stimulated ⁴⁵Ca²⁺ Uptake-- Previous work suggested that up-regulation of L-type channels by ethanol requires activation of PKC since it is prevented by the nonselective PKC inhibitors sphingosine and polymyxin B (29). Since we had used a different clone of PC12 cells for these earlier studies, we needed to determine whether our current PC12 cell line responds similarly to ethanol. In control cells, ⁴⁵Ca²⁺ uptake was 3.6 ± 0.21 nmol of Ca²⁺/mg of protein/2.5 min (n = 103) and was inhibited 92 ± 2% by the L-type channel antagonist nimodipine (1 µM). Exposure to 25-150 mM ethanol for 1-6 days increased K⁺-stimulated ⁴⁵Ca²⁺ uptake assayed in the absence of ethanol (Fig. 1 A and B). This is similar to the increase we observed previously with another PC12 cell line (8), except that a maximal response was achieved at 6 days in those cells instead of at 5 days as observed in the current cell line. To examine whether the increase in K⁺-stimulated ⁴⁵Ca²⁺ uptake was PKC-dependent, we cultured cells in the presence of 1 µM GF 109203X, which is a relatively selective PKC inhibitor that inhibits all phorbol ester-sensitive PKC isozymes except PKCµ (37, 38). As shown in Fig. 1A, GF 109203X prevented increases in K⁺-stimulated ⁴⁵Ca²⁺ uptake induced by treatment with 150 mM ethanol for 6 days. Thus, ethanol appears to increase K⁺-stimulated ⁴⁵Ca²⁺ uptake in these cells by a PKC-dependent mechanism.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Time course and concentration dependence of increases in K+-stimulated 45Ca2+ uptake following exposure to ethanol. A, 45Ca2+ uptake was measured in PC12 cells cultured with 150 mM ethanol (). Some cultures were treated with 1 µM GF 109203X with () or without () ethanol. B, 45Ca2+ uptake was measured in PC12 cells cultured for 6 days in the indicated concentrations of ethanol. The data shown are the means ± S.E. (n = 3-16) and are expressed as the percent above 45Ca2+ uptake measured in parallel control cells cultured without ethanol.

Characterization of V1-expressing Cell Lines-- To investigate whether PKC or PKC is required for up-regulation of L-type calcium channels by ethanol, we created PC12 cell lines that stably express V1 fragments derived from the first variable domains of PKC (V1) and PKC (V1). These fragments inhibit phorbol ester-induced translocation and activation of their corresponding PKC isozyme (32, 33). Characterization of V1-expressing PC12 cell lines (V11 and V12) was described recently (33). Expression of the V1 fragment in V12, V13, and V14 cells was confirmed by reverse transcriptase-polymerase chain reaction (Fig. 2A) and Western analysis (Fig. 2B). Treatment with 30 nM PMA stimulated translocation of both PKC and PKC to the particulate fraction in the parent PC12 cell line and in C cells transfected with vector alone (Fig. 2, C and D). However, in cell lines expressing the V1 fragment, translocation of PKC was inhibited, whereas translocation of PKC was not (Fig. 2, C and D). In contrast, PMA-induced translocation of PKC is selectively inhibited in V1-expressing lines (33). Thus, expression of V1 or V1 fragments selectively inhibits phorbol ester-stimulated translocation of the corresponding PKC isozyme in these cells.

View larger version (39K): [in this window] [in a new window]   Fig. 2.   Characterization of stably transfected PC12 cells expressing Flag epitope-tagged V1. A, reverse transcriptase-polymerase chain reaction products were analyzed on 1.2% agarose gels to demonstrate expression of V1-Flag mRNA in clones V12, V13, and V14. No product was found in PC12 cells or C cells transfected with vector alone. kb, kilobase; Std, DNA size markers. B, lysates of V12, V13, and V14 cells were analyzed on Western blots to detect the presence of 17-kDa Flag immunoreactivity that comigrates with purified Flag-tagged V1 expressed in bacteria (-Flag). C, cells were treated with 30 nM PMA for 2 min to induce PKC translocation and then fractionated into cytosolic (Cy) and particulate (P) cell fractions. PKC and PKC immunoreactivity in cell fractions was detected by Western analysis. The data shown are from a representative experiment. D, PKC isozyme-specific immunoreactivity on Western blots of cytosolic and particulate fractions was quantified by scanning densitometry and is expressed as a percentage of total immunoreactivity. PKC isozyme translocation was then calculated as the increase in isozyme-specific immunoreactivity found in the particulate fraction following treatment with PMA. The data shown are the means ± S.E. from four to seven experiments. *, p < 0.01 compared with PC12 or C cells (ANOVA and Newman-Keuls test).

The V1 Fragment Prevents Ethanol-induced Increases in K⁺-stimulated ⁴⁵Ca²⁺ Uptake-- To determine whether PKC or PKC mediates ethanol-induced increases in L-type channel function, we measured K⁺-stimulated ⁴⁵Ca²⁺ uptake in cells expressing V1 or V1. Expressed as a percentage of K⁺-stimulated ⁴⁵Ca²⁺ uptake measured in PC12 cells, uptake was similar (p = 0.11; ANOVA) in C (88 ± 6%, n = 23), V12 (107 ± 10%, n = 15), V13 (118 ± 7%, n = 6), V14 (85 ± 6%, n = 8), V11 (101 ± 7%, n = 6), and V12 (98 ± 5%, n = 6) cells. Treatment with 150 mM ethanol for 6 days increased ⁴⁵Ca²⁺ uptake in PC12, C, and V1-expressing cells to a similar extent (Fig. 3A). In contrast, ethanol was much less effective in increasing ⁴⁵Ca²⁺ uptake in cells expressing the V1 fragment (Fig. 3A).

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Ethanol-induced increases in 45Ca2+ uptake and dihydropyridine binding are inhibited in cells expressing V1. Cells were treated with 150 mM ethanol for 6 days. A, depolarization-induced 45Ca2+ uptake was measured in PC12 cells, vector-transfected cells (C), and cells expressing the V1 fragment (V12, V13, or V14) or the V1 fragment (V11 or V12). The data shown are the means ± S.E. (n = 5-24) and are expressed as the percent above 45Ca2+ uptake measured in parallel control cells cultured without ethanol. *, p < 0.05 compared with PC12 or C cells (ANOVA and Newman-Keuls test). B, binding of (+)-[3H]PN200-110 was measured in depolarized cells, and the results are expressed as the percent above or below specific binding measured in parallel control cells cultured without ethanol. The data shown are the means ± S.E. (n = 6-9). *, p < 0.05 compared with PC12 or C cells (ANOVA and Newman-Keuls test).

The V1 Fragment Prevents Ethanol-induced Increases in Dihydropyridine Binding-- To determine if expression of the V1 fragment also prevents ethanol-induced increases in dihydropyridine binding, we measured binding of the L-type calcium channel antagonist (+)-[³H]PN200-110 to ethanol-treated cells (36). For these studies, we selected the two V1-expressing clones in which the response to ethanol was most inhibited in the ⁴⁵Ca²⁺ uptake assay. Basal (+)-[³H]PN200-110 binding to PC12 cells was 3.52 ± 0.43 fmol/mg (n = 15) and was similar (p = 0.14; ANOVA) to binding measured in C (4.43 ± 0.42 fmol/mg, n = 7), V12 (3.78 ± 0.64 fmol/mg, n = 8), V13 (4.85 ± 0.49 fmol/mg, n = 7), V11 (3.17 ± 0.32 fmol/mg, n = 8), and V12 (3.17 ± 0.37 fmol/mg, n = 6) cells. Treatment with 150 mM ethanol for 6 days increased binding to a similar extent in PC12, C, V11, and V12 cells (Fig. 3B). In contrast, ethanol failed to increase binding in V12 and V13 cells. These results suggest that PKC is required for up-regulation of L-type channels by ethanol.

Overexpression of PKC-- To examine whether increases in PKC are sufficient to increase L-channel density, we examined (+)-[³H]PN200-110 binding in PC12 cell lines 1 and 2, which overexpress PKC (34). Compared with nontransfected PC12 cells, binding of 50 pM (+)-[³H]PN200-110 was increased in 1 and 2 cells, but not in C cells transfected with vector alone (Fig. 4). To determine whether the increase in binding in PKC-overexpressing cells was due to a change in binding affinity or binding site number, binding was measured in 2 cells at increasing concentrations of (+)-[³H]PN200-110 (from 10 to 280 pM). Fig. 5 shows a representative experiment. Scatchard analysis yielded similar values for the equilibrium dissociation constant (K_D) in PC12 (84 ± 4 pM) and 2 (102 ± 14 pM) cells (p = 0.27; n = 3). In contrast, the maximal number of binding sites (B_max) in 2 cells (16.8 ± 1.9 fmol/mg) was greater (p < 0.028; n = 3) than the B_max in PC12 cells (10.0 ± 0.7 fmol/mg). These findings are consistent with an increase in L-channel density rather than an increase in binding affinity in cells that overexpress PKC. However, despite the increase in dihydropyridine binding, K⁺-stimulated ⁴⁵Ca²⁺ uptake was not increased in 1 or 2 cells (Fig. 4).

View larger version (17K): [in this window] [in a new window]   Fig. 4.   K+-stimulated 45Ca2+ uptake and dihydropyridine binding in cells that overexpress PKC. K+-stimulated 45Ca2+ uptake (gray bars) and binding of 50 pM (+)-[3H]PN200-110 (black bars) were measured in 1 and 2 cells that overexpress rat PKC and in C cells transfected with vector alone. The data shown are the means ± S.E. from 3 to 19 experiments and are expressed as the percent of uptake and binding above or below that measured in parallel cultures of PC12 cells. *, p < 0.016 by one-sample t test.

View larger version (15K): [in this window] [in a new window]   Fig. 5.   Saturation analysis of (+)-[3H]PN200-110 specific binding in PC12 and 2 cells. Data from saturation isotherms (A) were converted to Scatchard plots (B), and values for KD (1/slope) and Bmax (x intercept) were determined by linear regression analysis. The data shown are from a representative experiment performed in triplicate. Mean KD and Bmax ± S.E. from three experiments are given under "Results."

PKC-independent Regulation of L-type Channels by Ethanol-- Since overexpression of PKC increased dihydropyridine binding but not K⁺-stimulated ⁴⁵Ca²⁺ uptake, we considered whether enhancement of L-channel function by ethanol requires additional, PKC-independent mechanisms. Initial evidence for a PKC-independent mechanism was obtained by examining V1-expressing cells treated with ethanol and PKC inhibitors. Although treatment with GF 109203X substantially inhibited ethanol-induced increases in K⁺-stimulated ⁴⁵Ca²⁺ uptake in PC12 cells (Fig. 1A), in V14 cells, it did not prevent increases due to ethanol, which were 26 ± 3% of control in the absence and 25 ± 5% of control in the presence of GF 109203X (p = 0.9; n = 6). This result indicates that a PKC-independent mechanism activated by ethanol contributes to L-channel up-regulation. To examine if such a PKC-independent mechanism is required to increase L-channel function in PKC-overexpressing cells, we treated 1 and 2 cells with 150 mM ethanol for 5 days. We predicted that this treatment would markedly increase ⁴⁵Ca²⁺ uptake with little or no effect on dihydropyridine binding. Ethanol enhanced K⁺-stimulated ⁴⁵Ca²⁺ uptake in these cells (Fig. 6A) without increasing PKC immunoreactivity (Fig. 6B), consistent with activation of a PKC-independent mechanism. However, the increase in ⁴⁵Ca²⁺ uptake was modest and associated with a similar increase in dihydropyridine binding (Fig. 6A).

View larger version (28K): [in this window] [in a new window]   Fig. 6.   Ethanol treatment of PKC-overexpressing cells. Cells were treated with 150 mM ethanol for 5 days. A, K+-stimulated 45Ca2+ uptake (stippled bars) and binding of 50 pM (+)-[3H]PN200-110 (black bars) were measured in PC12 cells and in 1 and 2 cells that overexpress PKC. The data shown are the means ± S.E. from three to eight uptake experiments and four binding experiments and are expressed as the percent of uptake or binding above that measured in parallel control cultures of each cell line treated without drugs. Ethanol-induced increases in 45Ca2+ uptake and dihydropyridine binding in 1 and 2 cells were significantly less than in PC12 cells (p < 0.05; ANOVA and Newman-Keuls test). B, shown is the PKC immunoreactivity on Western blots of total cell lysates (mean ± S.E., n = 2). Inset, Western blot of control (C) and ethanol-treated (E) cells from a representative experiment. The data shown are expressed as the percent of PKC immunoreactivity above or below that measured in parallel control cultures of each cell line treated without ethanol.

	DISCUSSION

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In this paper, we found that ethanol-induced increases in L-channel density and function were largely prevented by expression of V1, a selective inhibitor of PKC translocation. In contrast, expression of V1, which inhibits PKC translocation, did not prevent L-channel up-regulation. Expression of V1 did not alter L-channel density or function in the absence of ethanol. These results indicate that PKC, but not PKC, is important for ethanol-induced increases in functional L-type calcium channels in PC12 cells, but not for the basal activity of these channels.

Since ethanol increases the abundance of PKC (30), we examined whether overexpression of PKC would mimic the effect of ethanol on L-channel density and function. Although PKC overexpression increased the number of dihydropyridine-binding sites in PC12 cells, it did not increase K⁺-stimulated ⁴⁵Ca²⁺ uptake. This could have occurred because PKC-independent mechanisms are also required to increase channel function. Two results provided evidence for PKC-independent mechanisms that are activated by ethanol. In V14 cells, PKC translocation was completely blocked with only partial suppression of the response to ethanol, and the remaining ethanol-induced response was resistant to the PKC inhibitor GF 109203X. Moreover, in PKC-overexpressing cells, ethanol increased K⁺-stimulated ⁴⁵Ca²⁺ uptake and dihydropyridine binding without a further increase in PKC immunoreactivity. These findings indicate that PKC-independent mechanisms contribute to up-regulation of L-type channels by ethanol.

If PKC-independent mechanisms activated by ethanol are required for increases in L-channel function, treatment of PKC-overexpressing cells with ethanol should have markedly increased ⁴⁵Ca²⁺ uptake to levels commensurate with levels of dihydropyridine binding in these cells. However, this did not occur. PKC overexpression alone increased dihydropyridine binding by ~1.6-fold (Fig. 4), and ethanol treatment by another 1.4-fold (Fig. 6), for a combined increase of 2.2-fold over binding in untreated PC12 cells. In contrast, PKC overexpression and ethanol treatment together only increased ⁴⁵Ca²⁺ uptake by 1.4-fold over uptake in untreated PC12 cells (Figs. 4 and 6). It appears that inactivity of PKC-independent mechanisms cannot explain why PKC overexpression alone increased L-channel density, but not L-channel function.

It is possible that increases in channel density evoked by stable overexpression of PKC activate homeostatic mechanisms that reduce channel function to maintain a set level of calcium signaling. These mechanisms could act by decreasing L-channel function through altered subunit composition or phosphorylation or by increasing calcium efflux through increased Na⁺-Ca²⁺ exchange and the action of membrane ATPases (39). Homeostatic mechanisms that regulate channel density are activated by treatments that alter L-channel activity. For example, prolonged treatment with drugs such as ethanol (8, 9, 14-16), morphine (40), or nifedipine (41), which inhibit L-channel activity, increases L-channel density. In contrast, prolonged activation by exposure to depolarizing concentrations of KCl (42) or the L-channel agonist Bay K8644 (43) decreases L-channel density. It appears that L-channel function is highly regulated, and any alteration in channel activity or density leads to compensatory responses that serve to normalize function in the continued presence of the perturbing stimulus. Further studies will be needed to identify these compensatory mechanisms and to determine if they are activated in PKC-overexpressing cells.

One mechanism by which ethanol, acting via PKC, could increase L-channel density is by increasing expression of channel subunits. Neuronal high voltage-activated calcium channels are multimeric complexes of at least three types of subunits: ₁, ₂, and (44). The major pharmacological and physiological features that distinguish different classes of voltage-gated channels are mainly due to ₁ subunits, which contain the calcium pore and binding sites for selective calcium channel antagonists. There are five genes known to encode ₁ subunits in brain (_1A, _1B, _1C, _1D, and _1E), and _1C and _1D are subunits of L-type channels (45-48). PC12 cells express _1C (49). Transfected _1C can form functional L-type channels, and coexpression with ₂ or  subunits results in increased channel function and a corresponding increase in dihydropyridine binding (47, 50-54). Thus, ethanol-induced increases in abundance of _1C, ₂, or  subunits could increase the number of functional L-type channels. This might occur at a transcriptional level since PKC activates AP-1/Jun-regulated gene expression (55, 56). Studies are currently underway to determine if chronic exposure to ethanol regulates expression of specific calcium channel subunits by a PKC-dependent mechanism.

PKC is ubiquitously expressed and has been implicated in control of cell growth (56-58), apoptosis (59), and exocytosis (60) in non-neuronal cells. Little is known about its role in neuronal cells. It is induced in rat brain through an N-methyl-D-aspartate receptor-dependent mechanism after transient focal ischemia (61), but its role in brain injury or repair is not known. PKC binds to the growth-associated protein GAP-43 and appears to act as a GAP-43 kinase (62). However, it is not yet clear if PKC specifically regulates functions such as neurite growth or neurotransmitter release, which appear to be modulated by GAP-43 (63, 64). Our results provide the first evidence of a functional role for PKC in neural cells. Our findings identify PKC as a regulator of L-channel density and a mediator of cellular adaptation to ethanol. Since L-type channels modulate drinking behavior (17-20) and contribute to manifestations of alcohol withdrawal (22-24), PKC may play a key role in alcohol consumption and dependence. Ongoing studies will determine if inhibition of PKC reduces ethanol consumption and the development of alcohol dependence in animals.