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• W2037664098 abstract "Chronic exposure to ethanol increases the number of functional L-type voltage-gated calcium channels in neural cells. In PC12 cells, this adaptive response is mediated by protein kinase C δ (PKCδ), but the mechanisms by which this occurs are not known. Since expression of several different calcium channel subunits can increase the abundance of functional L-type channels, we sought to identify which subunits are regulated by ethanol. Incubation of PC12 cells with 120–150 mm ethanol for 6 days increased levels of α1C, α2, and β1b subunit immunoreactivity in cell membranes and selectively increased the abundance of mRNA encoding the α1C-1 splice variant of α1C. In cells expressing a fragment of PKCδ (δV1) that selectively inhibits PKCδ, there was no increase in membrane-associated α1C, α2, and β1b immunoreactivity following chronic ethanol exposure. However, ethanol still increased levels of α1C-1 mRNA in these cells. These results indicate that ethanol increases the abundance of L-type channels by at least two mechanisms; one involves increases in mRNA encoding a splice variant of α1Cand the other is post-transcriptional, rate-limiting, and requires PKCδ. Chronic exposure to ethanol increases the number of functional L-type voltage-gated calcium channels in neural cells. In PC12 cells, this adaptive response is mediated by protein kinase C δ (PKCδ), but the mechanisms by which this occurs are not known. Since expression of several different calcium channel subunits can increase the abundance of functional L-type channels, we sought to identify which subunits are regulated by ethanol. Incubation of PC12 cells with 120–150 mm ethanol for 6 days increased levels of α1C, α2, and β1b subunit immunoreactivity in cell membranes and selectively increased the abundance of mRNA encoding the α1C-1 splice variant of α1C. In cells expressing a fragment of PKCδ (δV1) that selectively inhibits PKCδ, there was no increase in membrane-associated α1C, α2, and β1b immunoreactivity following chronic ethanol exposure. However, ethanol still increased levels of α1C-1 mRNA in these cells. These results indicate that ethanol increases the abundance of L-type channels by at least two mechanisms; one involves increases in mRNA encoding a splice variant of α1Cand the other is post-transcriptional, rate-limiting, and requires PKCδ. dihydropyridine protein kinase C bp, base pair polymerase chain reaction glyceraldehyde-3-phosphate dehydrogenase analysis of variance amino acids phosphate-buffered saline Voltage-gated calcium channels mediate calcium entry into neurons and regulate neurotransmitter release, firing patterns, gene expression, and differentiation (1Ghosh A. Greenberg M.E. Science. 1995; 268: 239-247Crossref PubMed Scopus (1240) Google Scholar, 2Dunlap K. Luebke J.I. Turner T.J. Trends Neurosci. 1995; 18: 89-98Abstract Full Text PDF PubMed Scopus (868) Google Scholar). L-type channels are a subfamily of voltage-gated calcium channels that are activated by high voltage, inactivate slowly, and are blocked by dihydropyridines (DHPs).1 Acute ethanol exposure inhibits the function of L-type channels in several neuronal preparations (3Mullikin-Kilpatrick D. Treistman S.N. J. Pharmacol. Exp. Ther. 1995; 272: 489-497PubMed Google Scholar, 4Wang X. Wang G. Lemos J.R. Treistman S.N. J. Neurosci. 1994; 14: 5453-5460Crossref PubMed Google Scholar, 5Wang X. Dayanithi G. Lemos J.R. Nordmann J.J. Treistman S.N. J. Pharmacol. Exp. Ther. 1991; 259: 705-711PubMed Google Scholar). In contrast, in the neural crest-derived cell line PC12, chronic ethanol exposure produces a reversible concentration- and time-dependent increase in K+-evoked45Ca2+ uptake and depolarization-evoked calcium currents through L-type channels (6Skattebol A. Rabin R. Biochem. Pharmacol. 1987; 36: 2227-2229Crossref PubMed Scopus (45) Google Scholar, 7Messing R.O. Carpenter C.L. Greenberg D.A. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 6213-6215Crossref PubMed Scopus (178) Google Scholar, 8Grant A.J. Koski G. Treistman S.N. Brain Res. 1993; 600: 280-284Crossref PubMed Scopus (36) Google Scholar). Ethanol-induced increases in L-type channel function are associated with corresponding increases in the density of binding sites for DHP calcium channel antagonists (6Skattebol A. Rabin R. Biochem. Pharmacol. 1987; 36: 2227-2229Crossref PubMed Scopus (45) Google Scholar,7Messing R.O. Carpenter C.L. Greenberg D.A. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 6213-6215Crossref PubMed Scopus (178) Google Scholar), indicating that chronic exposure to ethanol increases the number of functional L-type channels. Similar increases in DHP binding have been detected in brain membranes from ethanol-dependent rodents (9Dolin S. Little H. Hudspith M. Pagonis C. Littleton J. Neuropharmacology. 1987; 26: 275-279Crossref PubMed Scopus (159) Google Scholar, 10Guppy L.J. Crabbe J.C. Littleton J.M. Alcohol Alcohol. 1995; 30: 607-615PubMed Google Scholar). Up-regulation of L-type calcium channels appears to contribute to intense neuronal hyperexcitability observed during alcohol withdrawal since L channel antagonists reduce tremors, seizures, and mortality in alcohol-dependent rodents deprived of ethanol (11Little H.J. Dolin S.J. Halsey M.J. Life Sci. 1986; 39: 2059-2065Crossref PubMed Scopus (176) Google Scholar, 12Bone G.H. Majchrowicz E. Martin P.R. Linnoila M. Nutt D.J. Psychopharmacology. 1989; 99: 386-388Crossref PubMed Scopus (36) Google Scholar). Ethanol-induced increases in L-type channels may also promote alcohol consumption since L channel antagonists reduce ethanol self-administration in animals (13Fadda F. Garau B. Colombo G. Gessa G.L. Alcohol. Clin. Exp. Res. 1992; 16: 449-452Crossref PubMed Scopus (49) Google Scholar, 14Colombo G. Agabio R. Lobina C. Reali R. Fadda F. Gessa G.L. Eur. J. Pharmacol. 1994; 265: 167-170Crossref PubMed Scopus (15) Google Scholar, 15Rezvani A.H. Janowsky D.S. Prog. Neuropsychopharmacol. Biol. Psychiatry. 1990; 14: 623-631Crossref PubMed Scopus (45) Google Scholar, 16Rezvani A.H. Grady D.R. Janowsky D.S. Alcohol Alcohol. 1991; 26: 161-167Crossref PubMed Scopus (41) Google Scholar). Neuronal voltage-gated calcium channels are multimeric complexes of at least three types of subunits as follows: α1, α2δ, and β (2Dunlap K. Luebke J.I. Turner T.J. Trends Neurosci. 1995; 18: 89-98Abstract Full Text PDF PubMed Scopus (868) Google Scholar). Diversity within the α1subunit family is responsible for the major pharmacological and physiological features that distinguish the different classes of calcium channels. α1 subunits contain the calcium pore and binding sites for selective channel antagonists. They are comprised of four homologous repeats (I–IV) each containing six transmembrane segments (S1–S6). Four L-type channel α1 genes have been cloned thus far as follows: α1S from skeletal muscle (17Hogan K. Gregg R.G. Powers P.A. Genomics. 1996; 31: 392-394Crossref PubMed Scopus (26) Google Scholar), α1C from heart and brain (18Snutch T.P. Tomlinson W.J. Leonard J.P. M. G.M. Neuron. 1991; 7: 45-57Abstract Full Text PDF PubMed Scopus (294) Google Scholar), α1Dfrom neural and endocrine tissues (19Williams M.E. Feldman D.H. McCue A.F. Brenner R. Velicelebi G. Ellis S.B. Harpold M.M. Neuron. 1992; 8: 71-84Abstract Full Text PDF PubMed Scopus (438) Google Scholar), and α1F from retina (20Bech-Hansen N.T. Naylor M.J. Maybaum T.A. Pearce W.G. Koop B. Fishman G.A. Mets M. Musarella M.A. Boycott K.M. Nat. Genet. 1998; 19: 264-267Crossref PubMed Scopus (424) Google Scholar, 21Strom T.M. Nyakatura G. Apfelstedt-Sylla E. Hellebrand H. Lorenz B. Weber B.H. Wutz K. Gutwillinger N. Rüther K. Drescher B. Sauer C. Zrenner E. Meitinger T. Rosenthal A. Meindl A. Nat. Genet. 1998; 19: 260-263Crossref PubMed Scopus (394) Google Scholar). In brain, α1D and α1C are localized to neuronal cell bodies and proximal dendrites (22Hell J.W. Westenbroek R.E. Warner C. Ahijanian M.K. Prystay W. Gilbert M.M. Snutch T.P. Catterall W.A. J. Cell Biol. 1993; 123: 949-962Crossref PubMed Scopus (637) Google Scholar). Two splice variants of the rat α1C subunit have been identified, α1C-1 and α1C-2, and are differentially expressed in rat brain (18Snutch T.P. Tomlinson W.J. Leonard J.P. M. G.M. Neuron. 1991; 7: 45-57Abstract Full Text PDF PubMed Scopus (294) Google Scholar). The α1C-2protein differs from α1C-1 by having a 3-amino acid (aa) insert in the cytoplasmic loop between domains II and III and a 28-aa substitution in the S3 segment in domain IV. In the human α1C gene, this alternatively spliced IV-S3 transmembrane segment is encoded by homologous alternative exons 31 and 32 (23Soldatov N.M. Genomics. 1994; 22: 77-87Crossref PubMed Scopus (129) Google Scholar). It is not known if α1C-1 and α1C-2 differ in function. Protein kinase C (PKC) is a multigene family of phospholipid-dependent, serine-threonine kinases that regulate cell growth and differentiation, neurotransmitter release, receptor regulation, ion channel modulation, and gene expression (24Nishizuka Y. Science. 1992; 258: 607-614Crossref PubMed Scopus (4219) Google Scholar). Twelve PKC isozymes, encoded by 11 genes, have been identified (α, βI, βII, γ, δ, ε, ζ, η, θ, λ, μ, and ν) and differ in structure, requirements for activation, and patterns of expression (24Nishizuka Y. Science. 1992; 258: 607-614Crossref PubMed Scopus (4219) Google Scholar, 25Selbie L.A. Schmitz-Peiffer C. Sheng Y. Biden T.J. J. Biol. Chem. 1993; 268: 24296-24302Abstract Full Text PDF PubMed Google Scholar, 26Johannes F.-J. Prestle J. Eis S. Oberhagemann P. Pfizenmaier K. J. Biol. Chem. 1994; 269: 6140-6148Abstract Full Text PDF PubMed Google Scholar, 27Hayashi A. Seki N. Hattori A. Kozuma S. Saito T. Biochim. Biophys. Acta. 1999; 1450: 99-106Crossref PubMed Scopus (171) Google Scholar). We recently found that ethanol-induced increases in L-type channels can be blocked by an inhibitor of PKCδ (28Gerstin Jr., E.H. McMahon T. Hundle B. Dadgar J. Messing R.O. J. Biol. Chem. 1998; 273: 16409-16414Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). In this study we examined the abundance of specific calcium channel subunits and their mRNAs to explore further the mechanisms by which PKCδ mediates up-regulation of L-type channels by ethanol. Radioisotopes and nucleotides were purchased from Amersham Pharmacia Biotech. Restriction endonucleases and modifying enzymes were purchased from Promega (Madison, WI). JM109 (Promega) and XL-1 Blue (Stratagene, La Jolla, CA) bacteria were used. All other reagents were analytical grade and were from Sigma or Life Technologies, Inc. PC12 cells (J. Wagner, Cornell University) were cultured at 37 °C 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 120–150 mm ethanol in tightly capped flasks, and the medium was changed daily as in prior work (28Gerstin Jr., E.H. McMahon T. Hundle B. Dadgar J. Messing R.O. J. Biol. Chem. 1998; 273: 16409-16414Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Control samples were cultured in parallel without ethanol. For detection of α2 and β subunit immunoreactivity, cells were washed with PBS, solubilized in 1% digitonin, and homogenized in Buffer A containing 10 mmHEPES, pH 7.4, 0.3 m sucrose, 10 mm EDTA, 10 mm EGTA, and protease inhibitors (1 mmphenylmethylsulfonyl fluoride, 0.75 mm benzamide, 0.7 μg/ml pepstatin A, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 0.01 mg/ml lysozyme, and 8 mg/ml calpain 1 + 2). Cell membranes were collected by ultracentrifugation at 135,000 × g for 30 min at 4 °C and were resuspended in solubilization buffer containing 300 mm KCl, 150 mm NaCl, 10 mmsodium phosphate, pH 7.4, 10 mm EGTA, 10 mmEDTA, and the protease inhibitors used in Buffer A. Unsolubilized material was removed by centrifugation at 175,000 × gfor 45 min at 4 °C. For detection of α1C and neuronal γ (stargazin (29Letts V.A. Felix R. Biddlecome G.H. Arikkath J. Mahaffey C.L. Valenzuela A. Bartlett F.S., II Mori Y. Campbell K.P. Frankel W.N. Nat. Genet. 1998; 19: 340-347Crossref PubMed Scopus (482) Google Scholar)) subunit immunoreactivity, cells were treated as described (30Chien A.J. Zhao X. Shirokov R.E. Puri T.S. Chang C.F. Sun D. Rios E. Hosey M.M. J. Biol. Chem. 1995; 270: 30036-30044Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). Samples (80, 160, and 320 μg) of crude membrane pellet or digitonin-solubilized membranes were separated on SDS-polyacrylamide gels. Proteins were then transferred to nitrocellulose Hybond C Extra membranes (Amersham Pharmacia Biotech) for 2 h at 100 V, and membranes were incubated in PBS containing 0.1% Tween 20 and 5% nonfat dried milk for 1 h at room temperature. After incubation in primary antibody in the same buffer for 2 h at room temperature, or overnight at 4 °C, the blots were washed three times in PBS containing 0.1% Tween 20 for 10 min and then incubated with peroxidase-conjugated goat anti-rabbit IgG (Roche Molecular Biochemicals, 1:1000 dilution) for 1 h in PBS, 0.1% Tween 20 at room temperature. Calcium channel subunit immunoreactivity was detected by chemiluminescence with ECL reagent (Amersham Pharmacia Biotech). Immunoreactive bands were quantified by densitometric scanning, and linear regression analysis of protein concentrations and corresponding density values was used to determine the slope of the regression line for each condition. Slopes for ethanol-treated samples were divided by slopes calculated for paired control samples to calculate the percentage increase in immunoreactivity induced by ethanol. Polyclonal antibodies against α2, β1b, β2, β3, and neuronal γ (stargazin) subunits were obtained from K. Campbell (University of Iowa) and were used at dilutions of 1:500 to 1:1000. Polyclonal antibody CARD I against α1C (1:1000) was a gift from M. Hosey (Northwestern University). Antibody against α1D(1:600) was purchased from Alomone Labs (Jerusalem, Israel). Full-length rat α1C-2cDNA (a gift from T. Snutch, University of British Columbia; GenBankTM accession number M67515) was digested withBglII and PvuII, and a 498-bp cDNA fragment (nucleotides 3691–4188) that includes the mismatch between α1C-1 and α1C-2 in the IV S3 domain (nucleotides 4098–4181 of α1C-2) was subcloned into pSP72 (Promega). A BglII-linearized plasmid was used as a template to generate a [α-32P]CTP-labeled 498-bp cRNA probe using SP6 RNA polymerase. To generate a probe for α1C-1, two oligonucleotide primers were constructed from the rat α1C-1 sequence (GenBankTM accession number M67516) to flank the mismatch between α1C-1 and α1C-2 at the IV S3 domain as follows: JD1802 (CCCAAGCTTG GGTTTGACAA TGTTCTGGCA GCC), upstream of the mismatch with a HindIII site incorporated into the sequence, and JD1803 (CGCGGATCCG CGTGACGATG AGGAAGTCAA AAAC), downstream and spanning the mismatch region, with a BamHI site incorporated into the sequence. The first strand of α1C-1 cDNA was synthesized using the SuperScript Choice System for DNA synthesis kit (Life Technologies, Inc.). Total RNA (12 μg), isolated from PC12 cells, was heated to 70 °C for 10 min with 100 pmol of oligo(dT) primer (Life Technologies, Inc.) and then chilled on ice before 1× first strand buffer (Life Technologies, Inc.), 10 mm dithiothreitol, and 0.5 mm dNTP (dATP, dCTP, dGTP, dTTP) mix were added. The reaction was then heated for 2 min at 42 °C, and then 1 μl of SuperScript II reverse transcriptase (Life Technologies, Inc.) was added. After incubation for 1 h at 42 °C, 2 μl of the reaction product, containing reverse-transcribed cDNA, was added to a PCR mixture containing 1× polymerase chain reaction (PCR) buffer (Perkin-Elmer), 0.5 mm dNTP mix, 2.5 units of Amplitaq (Perkin-Elmer), 100 pmol of JD1802, and 100 pmol of JD1803. The reaction mixture was heated to 94 °C for 4 min and then subjected to 30 amplification cycles. Each cycle consisted of 53 °C for 45 s, 72 °C for 2 min, and 94 °C for 10 min. Finally, the mixture was incubated at 53 °C for 45 s and 2 °C for 2 min and then placed on ice. PCR products were digested with BamHI and HindIII and separated on a 1.2% agarose gel. The resultant fragments were excised and gel-purified using a QIAEX II Gel Extraction kit (Qiagen, Chatsworth, CA). Purified fragments were subcloned into pBluescript II SK(+) (Stratagene, La Jolla, CA), and positive colonies were sequenced. The sequence was identical to the predicted rat α1C-1sequence (nucleotides 4046–4628). A HindIII-linearized plasmid was used as a template with T3 RNA polymerase to generate a [α-32P]CTP-labeled 582-bp cRNA probe. Full-length rat β1b cDNA (a gift from E. Perez-Reyes, Loyola University, GenBankTM accession number X61394) was digested with NcoI and PstI, and the cDNA fragment (518 bp of coding sequence) was subcloned into pGEM5Zf(+) (Promega). An NcoI-linearized plasmid was used as a template with SP6 RNA polymerase to generate a [α-32P]CTP-labeled 518-bp probe. The plasmid pTRI-GAPDH-rat, which recognizes glyceraldehyde-3-phosphate dehydrogenase mRNA, was purchased from Ambion (Austin, TX) and used with T7 RNA polymerase to generate a [α-32P]CTP-labeled 316-bp probe. Total RNA was extracted from PC12 cells using the RNA STAT-60 method (Tel-Test, Friendswood, TX) and quantified by absorbance at 260 nm. Ribonuclease protection assays (RPAs) were performed as described previously (31Walter H.J. Berry M. Hill D.J. Cwyfan-Hughes J.M. Holly J.M.P. Logan A. Endocrinology. 1999; 140: 520-532Crossref PubMed Scopus (88) Google Scholar). Briefly, total RNA (20 μg) was dissolved in 30 μl of hybridization solution containing 60,000 cpm of a 32P-labeled calcium channel subunit cRNA probe and 10,000 cpm of a 32P-labeled GAPDH cRNA probe. The cRNA probes were allowed to anneal to the endogenous RNA at 45 °C overnight. The next day, digestion was performed at 37 °C for 30 min using an RNase solution containing a final concentration of 30 μg/ml RNase A (Ambion) and 800 units of RNase T1. The RNA:RNA hybrids were separated on a 5% polyacrylamide, 8m urea sequencing gel. The gel was dried, and mRNA fragments were visualized and densities calculated using a Storm 860 PhosphorImager and ImageQuant software (Molecular Dynamics). Century Template RNA markers (Ambion) were used to determine molecular weights. Results were expressed relative to parallel control samples cultured without ethanol. Total RNA (1, 2.5, 5, and 10 μg) was prepared for analysis in sample buffer (66% formamide, 10% formaldehyde, and 1× SSC). Samples were heated to 68 °C for 15 min before an equal volume of ice-cold 20× SSC was added. BA-85 nitrocellulose membranes were loaded into a slot blot apparatus (Schleicher & Schuell) and rinsed with 500 μl/well of 10× SSC. After the samples were loaded, the apparatus was washed with 1 ml of 10× SSC under vacuum. RNA was cross-linked to damp membranes under UV light. Plasmids containing cDNA inserts encoding α2δ (50 ng; a gift from A. Schwartz, University of Cincinnati) and GAPDH (50 ng; CLONTECH, Palo Alto, CA) were used as templates for random primer labeling using the Life Technologies, Inc., Radprime DNA labeling system. Excess radioactivity was removed by centrifugation through TE-10 columns (CLONTECH) at 700 ×g for 7 min. Membranes were placed in pre-hybridization solution (60% formamide, 3× SSC, 5× Denhardt's solution, 0.82 mm sodium pyrophosphate, 82 μg/ml salmon sperm DNA, 1% SDS) for 4 h at 42 °C. 32P-Labeled DNA (3–5 × 106 cpm/ml) was boiled for 10 min in the presence of salmon sperm DNA (0.1 mg/ml) and placed immediately on ice. Cooled32P-labeled probes were added to pre-hybridization solution with the addition of 5% w/v dextran sulfate and 1% SDS. After an overnight incubation at 42 °C, membranes were washed at 25 °C three times in 1× SSC, 0.1% SDS, 5 min for each wash. Radioactivity bound to the blots was quantified using a Storm 860 PhosphorImager and ImageQuant software. Levels of α2δ and GAPDH mRNA increased linearly over the concentrations of samples examined. Linear regression analysis of total mRNA and corresponding hybridization signals for α2δ and GAPDH mRNA were used to calculate the slope of the regression lines for each condition. Corresponding slopes calculated for α2δ and GAPDH mRNA were divided to normalize for abundance of GAPDH mRNA. Normalized slopes for ethanol-treated and paired control samples were then compared to calculate the percentage increase in α2δ mRNA induced by ethanol. To determine which L-type calcium channel subunits are regulated by ethanol, we first identified which subunits are expressed in PC12 cells. Western analysis of PC12 cell membranes with subunit-specific antibodies demonstrated immunoreactive bands of appropriate molecular mass (32Ellis S.B. Williams M.E. Ways N.R. Brenner R. Sharp A.H. Leung A.T. Campbell K.P. McKenna E. Koch W.J. Hui A. Schwartz A. Harpold M.M. Science. 1988; 241: 1661-1664Crossref PubMed Scopus (439) Google Scholar, 33Liu H. Felix R. Gurnett C.A. De Waard M. Witcher D.R. Campbell K.P. J. Neurosci. 1996; 16: 7557-7565Crossref PubMed Google Scholar) for α2 (140 kDa), β1b (72 kDa), and β3 (58 kDa) subunits (Fig. 1 A). We also found an 86-kDa immunoreactive band using an anti-β2 antibody (Fig. 1 A). In a prior report (33Liu H. Felix R. Gurnett C.A. De Waard M. Witcher D.R. Campbell K.P. J. Neurosci. 1996; 16: 7557-7565Crossref PubMed Google Scholar), this antibody labeled a 74-kDa protein in Western blots of PC12 cell membranes but recognized 87-, 74-, and 70-kDa protein bands in Western blots of rat cardiac microsome membranes. A 225-kDa band was detected using the anti-α1C antibody, which recognizes a band of 240 kDa in α1C-expressing Sf9 cells (34Puri T.S. Gerhardstein B.L. Zhao X.-L. Ladner M.B. Hosey M.M. Biochemistry. 1997; 36: 9605-9615Crossref PubMed Scopus (108) Google Scholar). Although we found α1D-like immunoreactivity in brain tissue, we were unable to detect immunoreactive bands of the appropriate molecular mass for this subunit in our line of PC12 cells (data not shown). This is similar to what has been previously reported by Liu and colleagues (33Liu H. Felix R. Gurnett C.A. De Waard M. Witcher D.R. Campbell K.P. J. Neurosci. 1996; 16: 7557-7565Crossref PubMed Google Scholar), who also found that PC12 cells do not express β4subunit immunoreactivity. We also did not detect immunoreactivity for the neuronal γ (stargazin) subunit in PC12 cells (data not shown). One mechanism by which ethanol could increase the density of functional L-type calcium channels in PC12 cells is by increasing the expression of α1 subunits, which form the calcium pore. However, α2 and especially β subunits promote the assembly and targeting of channel complexes to the cell membrane and together with α1C or α1D increase DHP binding and enhance L-type channel function (19Williams M.E. Feldman D.H. McCue A.F. Brenner R. Velicelebi G. Ellis S.B. Harpold M.M. Neuron. 1992; 8: 71-84Abstract Full Text PDF PubMed Scopus (438) Google Scholar, 35Shistik E. Ivanina T. Puri T. Hosey M. Dascal N. J. Physiol. (Lond.). 1995; 489: 55-62Crossref Scopus (126) Google Scholar, 36Hullin R. Singer-Lahat D. Freichel M. Biel M. Dascal N. Hofmann F. Flockerzi V. EMBO J. 1992; 11: 885-890Crossref PubMed Scopus (279) Google Scholar, 37Massa E. Kelly K.M. Yule D.I. Macdonald R.L. Uhler M.D. Mol. Pharmacol. 1995; 47: 707-716PubMed Google Scholar, 38Perez-Garcia M.T. Kamp T.J. Marban E. J. Gen. Physiol. 1995; 105: 289-306Crossref PubMed Scopus (75) Google Scholar, 39Nishimura S. Hiroshi T. Hofmann F. Flockerzi V. Imoto K. FEBS Lett. 1993; 324: 283-286Crossref PubMed Scopus (70) Google Scholar). Therefore, ethanol-induced increases in abundance of α2 or β subunits might also increase the density of functional L-type channels in PC12 cells. To investigate which calcium channel subunits were increased by ethanol, we examined subunit immunoreactivity in membranes isolated from PC12 cells treated with 150 mm ethanol for 0–6 days. Ethanol evoked a time-dependent increase in α1C immunoreactivity, with levels peaking at 6 days of ethanol exposure (Fig. 1, A and B). Ethanol also increased levels of immunoreactivity for α2 and β1b without altering levels of immunoreactivity for β2 or β3 subunits (Fig. 1, A andC). Ethanol did not induce the appearance of α1D-like immunoreactivity (data not shown). These findings suggest that in PC12 cells, chronic ethanol exposure increases the abundance of L-type calcium channels composed of α1C, α2, and β1b subunits. To examine the role of PKCδ in calcium channel regulation, we used PC12 cell lines that stably express the fragment δV1, which is derived from the first variable domain of PKCδ and selectively inhibits phorbol ester-induced translocation of PKCδ (40Johnson J.A. Gray M.O. Chen C.-H. Mochly-Rosen D. J. Biol. Chem. 1996; 271: 24962-24966Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar,41Hundle B. McMahon T. Dadgar J. Chen C.-H. Mochly-Rosen D. Messing R.O. J. Biol. Chem. 1997; 272: 15028-15035Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). No differences in basal levels of immunoreactivity for α1C, α2, and β1b were observed in PC12 cells, vector-transfected cells, or V1δ2 or V1δ3 cells that express δV1 (data not shown). Treatment with 150 mm ethanol for 6 days increased membrane-associated immunoreactivity for α1C, α2, and β1b by 60–75% in PC12 cells and vector-transfected cells (Fig. 2). However no increase was observed in V1δ2 or V1δ3 cells. These results suggest that although PKCδ does not regulate basal levels of α1C, α2, and β1b, it is required for ethanol-induced increases in these calcium channel subunits. Chronic exposure to ethanol alters the abundance of several proteins including tyrosine hydroxylase (42Gayer G.G. Gordon A. Miles M.F. J. Biol. Chem. 1991; 266: 22279-22284Abstract Full Text PDF PubMed Google Scholar) phosducin-like protein (43Miles M. Barhite S. Sganga M. Elliot M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10831-10835Crossref PubMed Scopus (87) Google Scholar), and the molecular chaperones Hsc 70 (44Miles M.F. Diaz J.E. DeGuzman V.S. J. Biol. Chem. 1991; 266: 2409-2414Abstract Full Text PDF PubMed Google Scholar), GRP 78 (45Miles M.F. Wilke N. Elliot M. Tanner W. Shah S. Mol. Pharmacol. 1994; 46: 873-879PubMed Google Scholar), and GRP 94 (45) by increasing gene expression. Ethanol-induced increases in L-type channels might also occur at a transcriptional level since PKCδ has been implicated in activation of AP1/Jun-regulated gene expression (46Hirai S. Izumi Y. Higa K. Kaibuchi K. Mizuno K. Osada S. Suzuki K. Ohno S. EMBO J. 1994; 13: 2331-2340Crossref PubMed Scopus (110) Google Scholar, 47Li W. Michieli P. Alimandi M. Lorenzi M.V. Wu Y. Wang L.-H. Heidaran M.A. Pierce J.H. Oncogene. 1996; 13: 731-737PubMed Google Scholar). To investigate this possibility, we measured the abundance of calcium channel subunit mRNAs in PC12 cells after chronic ethanol exposure. Levels of total RNA per cell were not altered by exposure to 120 mm ethanol for 1–5 days (n = 7,p = 0.3754, ANOVA). Therefore we performed all mRNA studies using total RNA. Subsequent ribonuclease protection assay (RPA) analysis of GAPDH mRNA expression demonstrated that GAPDH mRNA levels were not altered by exposure to 120 mm ethanol for 1–5 days (n = 4, p = 0.8742, ANOVA). Therefore, we used GAPDH as an internal control to normalize all subsequent RPAs and slot blots for variation in sample loading. These studies revealed that ethanol did not increase levels of mRNA for α2δ or β1b (Fig.3). Two alternatively spliced variants of rat α1C have been identified as α1C-1 and α1C-2 (18Snutch T.P. Tomlinson W.J. Leonard J.P. M. G.M. Neuron. 1991; 7: 45-57Abstract Full Text PDF PubMed Scopus (294) Google Scholar). The major sequence difference between these variants lies within the S3 segment of domain IV within a region that is 43% different at the nucleotide level (36 differences in 84 nucleotides). Therefore, to identify both splice variants simultaneously by RPA, we analyzed α1C mRNA using a riboprobe made from the domain IV S3 segment of α1C-2 that includes this region of mismatch at its 5′ end (Fig. 4 A). This riboprobe was predicted to protect a 498-bp fragment from α1C-2 mRNA. Mismatch within the 5′ tail of the riboprobe was predicted to yield a fragment that is 84 bp shorter (approximately 414 bp) when complexed with α1C-1mRNA. As expected, RPA analysis with this probe revealed two fragments, one approximately 500 bp and the other approximately 410 bp in size. Only the abundance of the shorter fragment was increased by chronic ethanol exposure (Fig. 4, B and C). Increases in this putative α1C-1 transcript were apparent within 1 day of ethanol exposure (p < 0.001, ANOVA, Newman Keuls) and persisted throughout the 6 days of treatment (p < 0.02, ANOVA, Newman Keuls). Upon removal of ethanol from the cultures, the abundance of this mRNA species declined rapidly, reaching base-line levels within 24 h. These findings suggest that chronic exposure to ethanol selectively increases the abundance of α1C-1 mRNA in PC12 cells. To ensure that increases in the 410-bp fragment observed with the α1C-2 probe represent increases in α1C-1mRNA, we repeated the RPA analysis with a probe made from α1C-1 cDNA. As predicted, this probe recognized a major fragment of approximately 582 bp, and exposure to 120 mm ethanol for 0–5 days increased its abundance (data not shown). The magnitude of this increase was similar to that observed for the 410-bp fragment detected with the α1C-2 probe. This finding confirms our results in Figs. 4 and5 suggesting that ethanol selectively increases levels of α1C-1 mRNA. If PKCδ mediates ethanol-induced increases in α1C-1 mRNA, then α1C-1 mRNA levels should not be altered in ethanol-treated cells that express δV1. To examine this possibility, we treated V1δ2 cells with 120 mm ethanol for 0–5 days and found that ethanol exposure produced a time-dependent increase in α1C-1 mRNA abundance (Fig. 5,A and B). This increase was also observed in a second δV1-expressing cell line V1δ3 (Fig. 5 C). In addition, increases in δV1-expresing cells were much greater than increases observed in PC12 or vector-transfected cells (Fig.5 C). Ethanol did not increase the abundance of α2δ or β1b mRNA in these δV1-expressing cells (Fig. 5, D and E). These results indicate that ethanol selectively increases α1C-1mRNA levels by a PKCδ-independent mechanism. Among the several calcium channel subunits that can contribute to the formation of L-type calcium channels, our clone of PC12 cells appears to express only α1C, α2, β1b, β2, and β3 subunits. In previous work we found that exposure of PC12 cells to 150 mm ethanol for 6 days increases DHP binding and the function of L-type calcium channels by 55–85% (28Gerstin Jr., E.H. McMahon T. Hundle B. Dadgar J. Messing R.O. J. Biol. Chem. 1998; 273: 16409-16414Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). In the current study, we found that exposure to the same concentration of ethanol for the same amount of time increases membrane-associated immunoreactivity for α1C, α2, and β1b calcium channel subunits by 60–75% without altering immunoreactivity for β2 or β3 subunits. Increases in α1C immunoreactivity followed a time course similar to that observed previously for increases in K+-stimulated45Ca2+ uptake in PC12 cells (7Messing R.O. Carpenter C.L. Greenberg D.A. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 6213-6215Crossref PubMed Scopus (178) Google Scholar, 28Gerstin Jr., E.H. McMahon T. Hundle B. Dadgar J. Messing R.O. J. Biol. Chem. 1998; 273: 16409-16414Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Since the magnitudes of ethanol-induced increases in DHP binding, L-type channel function, and α1C, α2, and β1b subunit immunoreactivities are similar, ethanol-induced increases L-type calcium channels are most likely due to increases in these calcium channel subunits. Recently we found that increases in DHP binding and L-type channel function following chronic ethanol exposure are inhibited in cells that express the selective PKCδ inhibitor, δV1 (28Gerstin Jr., E.H. McMahon T. Hundle B. Dadgar J. Messing R.O. J. Biol. Chem. 1998; 273: 16409-16414Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). In the current study, we found that ethanol-induced increases in α1C, α2, and β1b subunits are also completely blocked in δV1-expressing cells. Therefore, our present results are consistent with our previous findings and together indicate that PKCδ is required for ethanol-induced increases in the density of functional L-type channels in PC12 cells. Our finding that β1b, but not β2 or β3, was selectively increased by ethanol, suggests that ethanol, via PKCδ, specifically recruits β1b subunits to newly formed L-type channel complexes. Exposure to ethanol did not alter β1b mRNA, indicating that ethanol regulates the abundance of β1b by post-transcriptional mechanisms. β subunits contain consensus sites for PKC phosphorylation, and phosphorylation of β subunits by PKC has been proposed to regulate L-type channel function (48Castellano A. Wei X. Birnbaumer L. Perez-Reyes E. J. Biol. Chem. 1993; 268: 3450-3455Abstract Full Text PDF PubMed Google Scholar, 49Bouron A. Soldatov N.M. Reuter H. FEBS Lett. 1995; 377: 159-162Crossref PubMed Scopus (37) Google Scholar). Further studies will be required to determine whether PKCδ selectively phosphorylates and regulates β1b protein turnover or trafficking in ethanol-treated cells. Ethanol exposure also selectively increased mRNA for α1C-1 without altering levels of mRNA for the alternative splice variant, α1C-2, or for other channel subunits. This is the first report of ethanol regulating the abundance of a specific mRNA splice variant. The most striking difference between α1C-1 and α1C-2 is a 13-aa substitution within a 28-aa region corresponding to the S3 segment of transmembrane domain IV. Although most of the 13 substitutions within this region are conservative, one exception is the substitution of a proline in α1C-1 for an alanine in α1C-2 at the amino terminus of the S3 segment. Substitutions located within or near this region may regulate channel gating (50Bourinet E. Soong T.W. Sutton K. Slaymaker S. Mathews E. Monteil A. Zamponi G.W. Nargeot J. Snutch T.P. Nat. Neurosci. 1999; 2: 407-415Crossref PubMed Scopus (362) Google Scholar). Electrophysiological studies with expressed α1C-1 and α1C-2subunits will be required to investigate this possibility. Increases in α1C-1 were also observed in δV1-expressing cells, suggesting that ethanol increases levels of α1C-1mRNA by PKCδ-independent mechanisms. Neither α2δ nor β1b mRNA abundance was altered by ethanol treatment in these cell lines, indicating that the response is specific for α1C-1. Increases in α1C-1 mRNA could be due to ethanol-induced changes in the splicing of α1C transcripts leading to greater production of α1C-1 mRNA. Alternatively, ethanol may act to decrease α1C-1 mRNA degradation. Why these effects of ethanol should be specific for α1C1 mRNA is unknown and requires further study. In the parent PC12 cell line, α1C-1 mRNA levels were increased after 1 day of ethanol exposure and remained nearly constant as long as ethanol was present. In contrast, in δV1-expressing cells, ethanol induced a much greater rise in α1C-1 mRNA, which continued to increase over the 5 days of ethanol exposure. These results suggest that PKCδ normally acts to limit α1C-1mRNA abundance, possibly by regulating α1C mRNA splicing or by promoting degradation of α1C-1 mRNA. This could be due to a direct effect of PKCδ on α1CmRNA processing or might occur indirectly, if PKCδ-mediated increases in calcium channel proteins evoke homeostatic mechanisms that decrease α1C-1 mRNA abundance. Additional splice variants of human α1C have been identified (51Perez-Reyes E. Wei X. Castellano A. Birnbaumer L. J. Biol. Chem. 1990; 265: 20430-20436Abstract Full Text PDF PubMed Google Scholar). Alternative splicing of exons 21 and 22 of the human α1C gene produces splice variants in the S2 segment of transmembrane domain III, and these show differences in the voltage sensitivity of inhibition by DHP antagonists (52Soldatov N.M. Bouron A. Reuter H. J. Biol. Chem. 1995; 270: 10540-10543Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Alternative splicing in the cytoplasmic tail alters the kinetics and the calcium dependence of channel inactivation (53Soldatov N.M. Zühlke R.D. Bouron A. Reuter H. J. Biol. Chem. 1997; 272: 3560-3566Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Therefore, alternative splicing of α1C transcripts can confer distinct functional characteristics to neuronal L-type channels. Ongoing studies will investigate whether similar splice variants can be identified in rat neural cells and whether ethanol regulates their abundance. Our results provide additional evidence for PKCδ as a regulator of L-type channel density and a mediator of cellular adaptation to ethanol. Our findings also indicate that PKCδ acts via post-transcriptional mechanisms to increase the density of L-type channels. In addition, our results provide evidence for a PKCδ-independent mechanism leading to increases in α1C-1 mRNA that may also contribute to ethanol-induced up-regulation of L-type channels. However, PKCδ-dependent mechanisms appear to be essential and rate-limiting for increases in L-type channels, since inhibition of PKCδ completely prevents ethanol-induced increases in DHP binding and L-type channel function (28Gerstin Jr., E.H. McMahon T. Hundle B. Dadgar J. Messing R.O. J. Biol. Chem. 1998; 273: 16409-16414Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Since antagonists of L-type channels decrease alcohol self-administration (13Fadda F. Garau B. Colombo G. Gessa G.L. Alcohol. Clin. Exp. Res. 1992; 16: 449-452Crossref PubMed Scopus (49) Google Scholar, 15Rezvani A.H. Janowsky D.S. Prog. Neuropsychopharmacol. Biol. Psychiatry. 1990; 14: 623-631Crossref PubMed Scopus (45) Google Scholar, 16Rezvani A.H. Grady D.R. Janowsky D.S. Alcohol Alcohol. 1991; 26: 161-167Crossref PubMed Scopus (41) Google Scholar) and reduce manifestations of alcohol withdrawal (11Little H.J. Dolin S.J. Halsey M.J. Life Sci. 1986; 39: 2059-2065Crossref PubMed Scopus (176) Google Scholar, 12Bone G.H. Majchrowicz E. Martin P.R. Linnoila M. Nutt D.J. Psychopharmacology. 1989; 99: 386-388Crossref PubMed Scopus (36) Google Scholar, 54Littleton J.M. Little H.J. Whittington M.A. Psychopharmacology. 1990; 100: 387-392Crossref PubMed Scopus (95) Google Scholar, 55Colombo G. Agabio R. Lobina C. Reali R. Melis F. Fadda F. Gessa G.L. Alcohol Alcohol. 1995; 30: 125-131PubMed Google Scholar), PKCδ, through its actions on L-type channels, may play a key role in regulating alcohol consumption and dependence." @default.
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