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• W2034930415 abstract "We explored the involvement of protein kinase C (PKC) and its isoforms in the regulation of BNaC2. Reverse transcriptase PCR evaluation of PKC isoform expression at the level of mRNA revealed the presence of α and ε/ε′ in all glioma cell lines analyzed; most, but not all cell lines expressed δ and ζ. No messages were found for the βI and βII isotypes of PKC in the tumor cells. Normal astrocytes expressed β but not γ. The essential features of these results were confirmed at the protein level by Western analysis. This disproportionate pattern of PKC isoform expression in glioma cell lines was further echoed in the functional effects of these PKC isoforms on BNaC2 activity in bilayers. PKC holoenzyme or the combination of PKCβI and PKCβII isoforms inhibited BNaC2. Neither PKCε nor PKCζ or their combination had any effect on BNaC2 activity in bilayers. The inhibitory effect of the PKCβI and PKCβII mixture on BNaC2 activity was abolished by a 5-fold excess of a PKCε and PKCζ combination. PKC holoenzymes, PKCβI, PKCβII, PKCδ, PKCε, and PKCζ phosphorylated BNaC2in vitro. In patch clamp experiments, the combination of PKCβI and PKCβII inhibited the basally activated inward Na+ conductance. The variable expression of the PKC isotypes and their functional antagonism in regulating BNaC2 activity support the idea that the participation of multiple PKC isotypes contributes to the overall activity of BNaC2. We explored the involvement of protein kinase C (PKC) and its isoforms in the regulation of BNaC2. Reverse transcriptase PCR evaluation of PKC isoform expression at the level of mRNA revealed the presence of α and ε/ε′ in all glioma cell lines analyzed; most, but not all cell lines expressed δ and ζ. No messages were found for the βI and βII isotypes of PKC in the tumor cells. Normal astrocytes expressed β but not γ. The essential features of these results were confirmed at the protein level by Western analysis. This disproportionate pattern of PKC isoform expression in glioma cell lines was further echoed in the functional effects of these PKC isoforms on BNaC2 activity in bilayers. PKC holoenzyme or the combination of PKCβI and PKCβII isoforms inhibited BNaC2. Neither PKCε nor PKCζ or their combination had any effect on BNaC2 activity in bilayers. The inhibitory effect of the PKCβI and PKCβII mixture on BNaC2 activity was abolished by a 5-fold excess of a PKCε and PKCζ combination. PKC holoenzymes, PKCβI, PKCβII, PKCδ, PKCε, and PKCζ phosphorylated BNaC2in vitro. In patch clamp experiments, the combination of PKCβI and PKCβII inhibited the basally activated inward Na+ conductance. The variable expression of the PKC isotypes and their functional antagonism in regulating BNaC2 activity support the idea that the participation of multiple PKC isotypes contributes to the overall activity of BNaC2. The recent molecular identification of a class of proton-sensitive ion channels (ASIC; acid-sensitiveion channel (1Waldmann R. Lazdunski M. Curr. Opin. Neurobiol. 1998; 8: 418-424Crossref PubMed Scopus (445) Google Scholar, 2Waldmann R. Champigny G. Lingueglia E. De Weille J.R. Heurteaux C. Lazdunski M. Ann. N. Y. Acad. Sci. 1999; 868: 67-76Crossref PubMed Scopus (180) Google Scholar); also called BNC and BNaC;brain Na+channel (3Price M.P. Snyder P.M. Welsh M.J. J. Biol. Chem. 1996; 271: 7879-7882Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar,4Garcı́a-Añoveros J. Derfler B. Neville-Golden J. Hyman B.T. Corey D.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1459-1464Crossref PubMed Scopus (296) Google Scholar)) belonging to the degenerin (DEG)/ENaC superfamily of ion channels (5Corey D.P. Garcı́a-Añoveros J. Science. 1996; 273: 323-324Crossref PubMed Scopus (101) Google Scholar) added a new molecular entity to the already complicated field of nociception. Even though their participation in nociception is controversial, ASICs might underlie some properties of native proton-induced currents (6Krishtal O.A. Pidoplichko V.I. Neuroscience. 1980; 12: 2325-2327Crossref Scopus (381) Google Scholar, 7Krishtal O.A. Pidoplichko V.I. Neuroscience. 1981; 24: 243-246Google Scholar, 8Bevan S. Yeats J. J. Physiol. 1991; 433: 145-161Crossref PubMed Scopus (302) Google Scholar) and could contribute to the function of nociceptive transduction with many other key constituents including nociceptor-specific voltage-gated Na+ channels, ATP-gated channels, and capsaicin receptors (9Wood J.N. Docherty R. Annu. Rev. Physiol. 1997; 59: 457-482Crossref PubMed Scopus (88) Google Scholar, 10McCleskey E.W. Gold M.S. Annu. Rev. Physiol. 1999; 61: 835-856Crossref PubMed Scopus (293) Google Scholar, 11Kress M. Zeilhofer H.U. Trends Pharmacol. Sci. 1999; 20: 112-118Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 12Caterina M.J. Julius D. Curr. Opin. Neurobiol. 1999; 9: 525-530Crossref PubMed Scopus (154) Google Scholar, 13Reeh P.W. Kress M. Curr. Opin. Pharmacol. 2001; 1: 45-51Crossref PubMed Scopus (140) Google Scholar). The distinguishing feature of this family of ion channels, namely, proton-induced conductance, is exhibited by ASIC1a (14Waldmann R. Champigny G. Bassilana F. Heurteaux C. Lazdunski M. Nature. 1997; 386: 173-177Crossref PubMed Scopus (1148) Google Scholar), BNaC2 (4Garcı́a-Añoveros J. Derfler B. Neville-Golden J. Hyman B.T. Corey D.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1459-1464Crossref PubMed Scopus (296) Google Scholar), ASIC1b (15Bässler E.L. Ngo-Anh T.J. Geisler H.S. Ruppersberg J.P. Gründer S. J. Biol. Chem. 2001; 276: 33782-33787Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar), ASICβ (16Chen C.C. England S. Akopian A.N. Wood J.N. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10240-10245Crossref PubMed Scopus (401) Google Scholar), ASIC2 (or BNC1) (3Price M.P. Snyder P.M. Welsh M.J. J. Biol. Chem. 1996; 271: 7879-7882Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar), BNaC1 (4Garcı́a-Añoveros J. Derfler B. Neville-Golden J. Hyman B.T. Corey D.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1459-1464Crossref PubMed Scopus (296) Google Scholar), MDEG (17Waldmann R. Champigny G. Voilley N. Lauritzen I. Lazdunski M. J. Biol. Chem. 1996; 271: 10433-10436Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar), and ASIC3 (DRASIC; 18), hASIC3 (19de Weille J.R. Bassilana F. Lazdunski M. Waldmann R. FEBS Lett. 1998; 433: 257-260Crossref PubMed Scopus (108) Google Scholar, 20Babinski K. Le K.T. Seguela P. J. Neurochem. 1999; 72: 51-57Crossref PubMed Scopus (159) Google Scholar), and hTNaC1 (21Ishibashi K. Marumo F. Biochem. Biophys. Res. Commun. 1998; 245: 589-593Crossref PubMed Scopus (45) Google Scholar). However, ASIC4 is functionally inactive (22Akopian A.N. Chen C.C. Ding Y. Cesare P. Wood J.N. Neuroreport. 2000; 11: 2217-2222Crossref PubMed Scopus (182) Google Scholar, 23Gründer S. Geissler H.S. Bässler E.L. Ruppersberg J.P. Neuroreport. 2000; 11: 1607-1611Crossref PubMed Scopus (197) Google Scholar) and may require an association with an accessory protein(s) and/or other subunit(s) of the family, like the splice variant form of ASIC2 (ASIC2b, also called MDEG2) (24Lingueglia E. de Weille J.R. Bassilana F. Heurteaux C. Sakai H. Waldmann R. Lazdunski M. J. Biol. Chem. 1997; 272: 29778-29783Abstract Full Text Full Text PDF PubMed Scopus (440) Google Scholar) or ASICβ2, a splice variant of ASICβ (25Ugawa S. Ueda T. Takahashi E. Hirabayashi Y. Yoneda T. Komai S. Shimada S. Neuroreport. 2001; 12: 2865-2869Crossref PubMed Scopus (29) Google Scholar). The multiplicity of current responses to extracellular acid loads in different neurons is consistent with the existence of functionally distinct ASICs in these cells. The tissue distribution of the ASIC members is not limited to the nervous system but also includes many other tissues such as the lung, testis, and intestine (20Babinski K. Le K.T. Seguela P. J. Neurochem. 1999; 72: 51-57Crossref PubMed Scopus (159) Google Scholar, 21Ishibashi K. Marumo F. Biochem. Biophys. Res. Commun. 1998; 245: 589-593Crossref PubMed Scopus (45) Google Scholar, 26Gunthorpe M.J. Smith G.D. Davis J.B. Randall A.D. Pflugers Arch. 2001; 442: 668-674Crossref PubMed Scopus (101) Google Scholar, 27Yiangou Y. Facer P. Smith J.A. Sangameswaran L. Eglen R. Birch R. Knowles C. Williams N. Anand P. Eur. J. Gastroenterol. Hepatol. 2001; 13: 891-896Crossref PubMed Scopus (112) Google Scholar). Sensory neuron-specific expression of DRASIC has been reported (18Waldmann R. Bassilana F. de Weille J. Champigny G. Heurteaux C. Lazdunski M. J. Biol. Chem. 1997; 272: 20975-20978Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar), but Chen et al.(16Chen C.C. England S. Akopian A.N. Wood J.N. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10240-10245Crossref PubMed Scopus (401) Google Scholar) have found low level transcripts in superior cervical ganglia, spinal cord, and brain stem. ASICs have been characterized extensively in heterologous expression systems, and besides being implicated in nociception (1Waldmann R. Lazdunski M. Curr. Opin. Neurobiol. 1998; 8: 418-424Crossref PubMed Scopus (445) Google Scholar, 2Waldmann R. Champigny G. Lingueglia E. De Weille J.R. Heurteaux C. Lazdunski M. Ann. N. Y. Acad. Sci. 1999; 868: 67-76Crossref PubMed Scopus (180) Google Scholar), a role in mechanotransduction (28Price M.P. Lewin G.R. McIlwrath S.L. Cheng C. Xie J. Heppenstall P.A. Stucky C.L. Mannsfeldt A.G. Brennan T.J. Drummond H.A. Qiao J. Benson C.J. Tarr D.E. Hrstka R.F. Yang B. Williamson R.A. Welsh M.J. Nature. 2000; 407: 1007-1011Crossref PubMed Scopus (425) Google Scholar, 29Garcı́a-Añoveros J. Samad T.A. Woolf C.J. Corey D.P. J. Neurosci. 2001; 21: 2678-2686Crossref PubMed Google Scholar), in the cellular response to an ischemic offense (27Yiangou Y. Facer P. Smith J.A. Sangameswaran L. Eglen R. Birch R. Knowles C. Williams N. Anand P. Eur. J. Gastroenterol. Hepatol. 2001; 13: 891-896Crossref PubMed Scopus (112) Google Scholar, 30Biagini G. Babinski K. Avoli M. Marcinkiewicz M. Seguela P. Neurobiol. Dis. 2001; 8: 45-58Crossref PubMed Scopus (68) Google Scholar, 31Johnson M.B. Jin K.L. Minami M. Chen D. Simon R.P. J. Cereb. Blood Flow Metab. 2001; 21: 734-740Crossref PubMed Scopus (79) Google Scholar), and synaptic plasticity (32Wemmie J.A. Chen J. Askwith C.C. Hruska-Hageman A.M. Price M.P. Nolan B.C. Yoder P.G. Lamani E. Hoshi T. Freeman J.H. Welsh M.J. Neuron. 2002; 34: 463-477Abstract Full Text Full Text PDF PubMed Scopus (556) Google Scholar, 33Bianchi L. Driscoll M. Neuron. 2002; 34: 337-340Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar), have been proposed. Despite significant characterization of ASICs, the possible role of second messenger regulation of ASICs has not been reported. Moreover, Bubien et al. (34Bubien J.K. Keeton D.A. Fuller C.M. Gillespie G.Y. Reddy A.T. Mapstone T.B. Benos D.J. Am. J. Physiol. 1999; 276: C1405-C1410Crossref PubMed Google Scholar) reported an amiloride-sensitive Na+ current in malignant brain tumor cells and the presence of BNaC2 message in these cells. Also, human glioma cells show a differential expression of specific PKC 1The abbreviations used for: PKC, protein kinase C; RT, reverse transcriptase; MOPS, 4-morpholinepropanesulfonic acid; GBM, glioblastoma multiform. isoforms compared with normal astroglia (35Xiao H. Goldthwait D.A. Mapstone T. J. Neurosurg. 1994; 81: 734-740Crossref PubMed Scopus (52) Google Scholar). With this in mind, we explored the role of PKC and its isoforms in the regulation of BNaC2. We found 1) expression of PKCα, PKCε, PKCδ, and PKCζ in most cell lines and no expression of PKCβ in all glioma cell lines compared with normal astrocytes; 2) separately, PKCβI and PKCβII lacked a channel inhibitory effect, but in combination PKCβI and PKCβII inhibited channel activity in bilayers, which was comparable with the inhibitory effect of whole PKC; 3) PKCε and PKCζ individually and in combination did not inhibit BNaC2, but a 5-fold excess of a PKCε and PKCζ combination abolished the otherwise inhibitory influence of the PKCβI and PKCβII mixture; 4) whole PKC, PKCβI, PKCβII, PKCδ, PKCε, and PKCζ phosphorylated BNaC2 in vitro; 5) PKCβI plus PKCβII inhibited inward Na+ currents in human U87-MG glioma cells. Our findings of disproportionate expression of PKC isotypes in glioma cell lines and their antagonism with respect to influencing BNaC2 activity in bilayers suggest that different proportions of PKC isoforms differentially regulate BNaC2 activity. Also, dysregulation of BNaC2 resulting from an altered expression of the PKC isoforms could be responsible for an activated amiloride-sensitive Na+ current seen in glioma cells (34Bubien J.K. Keeton D.A. Fuller C.M. Gillespie G.Y. Reddy A.T. Mapstone T.B. Benos D.J. Am. J. Physiol. 1999; 276: C1405-C1410Crossref PubMed Google Scholar). Phospholipids were purchased from Avanti Polar Lipids (Alabaster, AL). PKC, PKC isoforms, and PKC inhibitor peptide 19–31 were purchased from Calbiochem. All other chemicals were reagent grade, and all solutions were made with distilled water and filter-sterilized before use (Sterivex-GS, 0.22 μm filter; Millipore Corp., Bedford, MA). Total RNA was isolated from human glioma cells and normal astrocytes using a modification of the method of Chomczynski and Sacchi (36Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63187) Google Scholar). The integrity of the RNA was verified after electrophoresis through 1% agarose-formaldehyde denaturing gels. One-step RT-PCR was performed to detect PKC isozyme mRNA with a Qiagen OneStep RT-PCR kit. Total reaction mixture was 50 μl, containing 0.2 μg RNA, 0.4 mm of each dNTP, 30 μm of forward and reverse primer, and appropriate OneStep RT-PCR enzyme mix and buffer. RT-PCR was carried out beginning with a single cycle of 50 °C for 30 min (reverse transcription), 95 °C for 15 min (initial PCR activation step), followed by cycles of 94 °C for 1 min, 56 °C for 1 min, and 72 °C for 1 min, for a total of 35 cycles. This was followed by a single cycle of 72 °C for 10 min to facilitate final extension. The primers utilized are listed in Table I.Table IPrimers for reverse transcriptase polymerase chain reaction (RT-PCR)GenePrimer typeaF, forward primer; R, reverse primer.bp positionPrimer sequenceProduct sizebpPKCαF1595–16145′-CCTATGGCGTCCTGTTGTAT-3′473R2048–20675′-GTTTGTTCTCGCTGGTGAGT-3′PKCβbRefers to common region of PKCβI and PKCβII.F1591–16115′-TTTGGCATGTGTAAGGAAAAC-3′376R1945–19665′-GTTCAAGTTTCTCCCAATCAAT-3′PKCδF1283–13025′-TTTCTCACCCACCTCATCTG-3′448R1709–17305′-CGAAGAGTTCATCCTCATCATC-3′PKCεF181–2005′-ACCAAGCAGAAGACCAACAG-3′421R582–6015′-TTCCTATGACACCCCAGATG-3′PKCγF1134–11535′-TGGTCCTTTGGAGTTCTGCT-3′410R1524–15435′-TAGGTGAAGCCCTGGAAATC-3′PKCζF952–9715′-AGTCGGTTGTTCCTGGTCAT-3′531R1462–14825′-CCTCTCTTTGGGGTCCTTATT-3′a F, forward primer; R, reverse primer.b Refers to common region of PKCβI and PKCβII. Open table in a new tab Primers were synthesized by Invitrogen. All primer sequences were searched in GenBankTM, and no similarities between primers and other human gene sequences were found except for the target gene we intended to amplify. To confirm this, we used full-length cDNAs (ATCC) for human PKC isoform α, β, γ, ε, and ζ as substrates in PCR reactions with each of the primer pairs. PCR products were analyzed by agarose gel electrophoresis and visualized by ethidium bromide staining. The primers we designed for each isoform generated amplicons only from the appropriate PKC isoform cDNA template and not from the other templates. Authenticity of each product was confirmed by size and digestion with three restriction enzymes, as well as by direct sequencing. Computer analysis of nucleotide and restriction enzyme mapping was done using the Genetics Computer Group Package (37Devereux J. Haeberli P. Smithies O. Nucleic Acids Res. 1984; 12: 387-395Crossref PubMed Scopus (11536) Google Scholar) on a Unix computer and were provided through the University of Alabama at Birmingham Center for AIDS Research. Human lymphocyte RNA preps (volunteer donors) were used as positive controls (data not shown; see Ref. 13Reeh P.W. Kress M. Curr. Opin. Pharmacol. 2001; 1: 45-51Crossref PubMed Scopus (140) Google Scholar). The protocol used for Western analyses for different PKC isoforms in glial tumor cell lines was identical to that described earlier (38Jovov B. Tousson A. Ji H.L. Keeton D. Shlyonsky V. Ripoll P.J. Fuller C.M. Benos D.J. J. Biol. Chem. 1999; 274: 37845-37854Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). The antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). In vitro phosphorylation by PKC and its isoforms was assayed by measuring the incorporation of [32P] into immunopurified protein (BNaC2) from [γ-32P]ATP, in a reaction mixture containing 20 mm Tris·HCl, pH 7.5, 10 mmMgCl2, 20 μm ATP, 15–50 kBq of [γ-32P]ATP, and 1 microunit of PKC or its isoforms. Immunopurification of in vitro translated protein was performed as described previously (38Jovov B. Tousson A. Ji H.L. Keeton D. Shlyonsky V. Ripoll P.J. Fuller C.M. Benos D.J. J. Biol. Chem. 1999; 274: 37845-37854Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). The incubation was carried out for 3 min at 30 °C, and the phosphorylated proteins were separated by SDS/PAGE and visualized by autoradiography. Where indicated, PKC activity was measured in the presence of 0.5 mmCaCl2 or 1 μm PKC inhibitor (peptide 19–31). Standard methods for oocyte isolation, cRNA preparation, and injection were used (39Awayda M.S. Ismailov I.I. Berdiev B.K. Benos D.J. Am. J. Physiol. 1995; 268: C1450-C1459Crossref PubMed Google Scholar). Oocyte membrane vesicles were prepared as described (39Awayda M.S. Ismailov I.I. Berdiev B.K. Benos D.J. Am. J. Physiol. 1995; 268: C1450-C1459Crossref PubMed Google Scholar, 40Perez G. Lagrutta A. Adelman J.P. Toro L. Biophys. J. 1994; 66: 1022-1027Abstract Full Text PDF PubMed Scopus (51) Google Scholar). Oocyte membrane vesicles were fused with planar lipid bilayers made of a 2:1 (w/w) diphytanoyl-phosphatidyl-ethanolamine/diphytanoyl-phosphatidylserine solution in n-octane (final lipid concentration 25 mg/ml). Bilayers were bathed with symmetrical 100 mm NaCl, 10 mm MOPS-Tris, 100 μm EGTA, 50 nm[Ca2+]free, pH 7.4. The Bound-and-Determined computer program was used to calculate the level of free [Ca2+] (41Brooks S.P. Storey K.B. Anal. Biochem. 1992; 201: 119-126Crossref PubMed Scopus (324) Google Scholar). Phosphorylation mixture contained 10 ng/ml of PKC or its isoforms, 5 μm diacylglycerol, 100 μm Mg-ATP. To verify the orientation of BNaC2 and its block by amiloride, at the end of each experiment 5 μmamiloride was added to the presumptive extracellular side of the channel. Single channel currents were measured using a conventional current-to-voltage converter with a 10 gigaohm feedback resistor (Eltec, Daytona Beach, FL) as described previously (42Ismailov I.I. Shlyonsky V.G. Alvarez O. Benos D.J. J. Physiol. 1997; 504: 287-300Crossref PubMed Scopus (15) Google Scholar). Single channel analyses were performed using pCLAMP 5.6 software (Axon Instruments, Burlingame, CA) on current records low pass-filtered at 300 Hz through an 8-pole Bessel filter (902 LPF; Frequency Devices, Haverhill, MA) prior to acquisition using a Digidata 1200 interface (Axon Instruments, Burlingame, CA). Whole-cell patch clamp experiments were performed on cultured human U87-MG glioma cells as described previously (34Bubien J.K. Keeton D.A. Fuller C.M. Gillespie G.Y. Reddy A.T. Mapstone T.B. Benos D.J. Am. J. Physiol. 1999; 276: C1405-C1410Crossref PubMed Google Scholar). PKC isoforms were included in the pipette solution at a final concentration of 5 ng/ml. RT-PCR using specific primer pairs for PKCα, PKCβ, PKCδ, PKCε, PKCγ, and PKCζ (Table I) was performed on total RNA isolated from SK-MG1 glioma cells (Fig.1 A, top). A similar analysis was carried out for primary cultures of human astrocytes, three first passage cultures of glioblastoma multiform (GBM) tumor resections (PT1, PT2, and PT3) and ten established cell lines, nine of which were originally derived from GBMs and one (D32GS) from a gliosarcoma (Fig. 1 A). These experiments revealed that PKCβ mRNA was expressed by normal astrocytes but not by any of the tumor cells. The astrocytes expressed all of the PKC isoforms examined, except for PKCγ. Only PKCα and PKCε/ε′ were detected in all of the gliomas. Likewise, PKCδ mRNA was expressed in all samples except D32GS. There was more variability in expression of PKCγ and PKCζ. These results demonstrated that PKCα, PKCε/ε′, and PKCδ were expressed reliably in all of the GBM glioma cell lines examined and that PKCβI and PKCβII were not expressed at all (the primer pairs used for the detection of PKCβ spanned the common region of PKCβI and PKCβII). Western blot analysis was also performed to examine protein expression of PKCβI, PKCε/ε′, and PKCζ in the astrocytes and SK-MG1 and U87-MG cells (Fig. 1 B). Similar to the results presented in Fig. 1 A, only astrocytes expressed PKCβI, all three cell types expressed PKCε/ε′, and the astrocytes and SK-MG1, but not U87-MG, expressed PKCζ. BNaC2 incorporated into planar lipid bilayers forms a functional amiloride-sensitive Na+ channel with a very low probability of being in an open state (PO ∼0.08) (43Berdiev B.K. Mapstone T.B. Markert J.M. Gillespie G.Y. Lockhart J. Fuller C.M. Benos D.J. J. Biol. Chem. 2001; 276: 38755-38761Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). However, buffering [Ca2+]free in the bilayer bathing solution to <100 nm significantly increases PO and thus provides the opportunity to investigate the effects of PKC and its isoforms on a wild-type active channel. Addition of the phosphorylation mixture (with holoenzyme PKC) to the bilayer bathing solution decreased BNaC2 PO from 0.89 ± 0.09 to 0.45 ± 0.06 without any effect on single channel conductance (Fig. 2 A). We next tested the hypothesis that specific PKC isoforms could affect BNaC2 activity in different ways. The rationale for this set of experiments was the following. First, PKC is a large family of related proteins with at least 11 isotypes, each with a distinctive primary structure, expression pattern, and subcellular localization (44Dekker L.V. Parker P.J. Trends Biochem. Sci. 1994; 19: 73-77Abstract Full Text PDF PubMed Scopus (920) Google Scholar). Second, several groups (35Xiao H. Goldthwait D.A. Mapstone T. J. Neurosurg. 1994; 81: 734-740Crossref PubMed Scopus (52) Google Scholar, 45Sharif T.R. Sharif M. Int. J. Oncol. 1999; 15: 237-243PubMed Google Scholar) have shown that PKCε/ε′ and PKCζ are overexpressed in many glial tumor cell lines, whereas PKCβ is reduced or even absent compared with normal human astrocytes (Fig.1 A). Third, amiloride-sensitive Na+ currents were observed in primary cultures of freshly resected tumors and established glioma cell lines (34Bubien J.K. Keeton D.A. Fuller C.M. Gillespie G.Y. Reddy A.T. Mapstone T.B. Benos D.J. Am. J. Physiol. 1999; 276: C1405-C1410Crossref PubMed Google Scholar) along with BNaC mRNA. We began by examining PKCβI and PKCβII effects on BNaC2 activity in bilayers. When added alone, neither PKCβI nor PKCβII had any effect on BNaC2 activity (Table II), but their combination significantly decreased BNaC2 PO from 0.91 ± 0.08 to 0.48 ± 0.06; no changes in single conductance were observed (Fig. 2 B). This inhibition of BNaC2 activity by the PKCβI and PKCβII combination was equivalent to that of whole PKC. These results support the hypothesis that PKCβI and PKCβII are essential, at least in planar lipid bilayers, for the inhibitory effect of PKC on BNaC2 activity. Because of the reported up-regulated levels of PKCε and PKCζ isotypes, and the expectation that these isoforms could have their own effects on BNaC2 activity, we explored the effect of PKCε and PKCζ on BNaC2 activity. We found that PKCε and PKCζ added alone or in combination, did not have any effect on BNaC2 activity (Table II). This outcome prompted us to imitate the differential levels of PKC isoform expression in gliomas in our bilayer experiments. A 5-fold excess of PKCε and PKCζ relative to PKCβI and PKCβII was added to the bilayer bathing solution following incorporation of BNaC2. This maneuver abolished the otherwise inhibitory effect of the PKCβI and PKCβII combination on BNaC2 activity in bilayers (Fig. 2 C). We did not observe this effect with a 1:1 ratio of isoforms (Table II). Also, a 5-fold excess of PKCδ or its combination with PKCε or PKCζ neither affected BNaC2 activity nor interfered with the inhibitory influence of the PKCβI and PKCβII combination on BNaC2 activity (Table II).Table IIThe effect of phosphorylation by PKC and its isoforms on properties of BNaC2 in planar lipid bilayersPKC or PKC isoform(s)Before phosphorylationAfter phosphorylationGPoGPopSpSPKC21 ± 2.00.89 ± 0.0921 ± 2.30.45 ± 0.06PKCβI19 ± 2.10.88 ± 0.0622 ± 1.80.92 ± 0.06PKCβII20 ± 1.90.91 ± 0.0619 ± 1.80.87 ± 0.08PKCβI + PKCβII23 ± 2.00.91 ± 0.0820 ± 2.10.48 ± 0.06PKCβI + PKCβII*21 ± 2.20.61 ± 0.0722 ± 1.80.24 ± 0.05PKCβI + PKCβII**19 ± 2.50.37 ± 0.0520 ± 2.10.07 ± 0.04PKCδ21 ± 1.90.90 ± 0.0620 ± 2.00.92 ± 0.06PKCε19 ± 2.80.87 ± 0.0821 ± 2.40.91 ± 0.06PKCζ21 ± 2.60.93 ± 0.0618 ± 2.70.93 ± 0.06PKCε + PKCζ19 ± 2.90.89 ± 0.0621 ± 1.60.88 ± 0.09PKCε + PKCζ:PKCβI + PKCβII (1:1)22 ± 1.80.92 ± 0.0720 ± 1.40.46 ± 0.07PKCε + PKCζ:PKCβI + PKCβII (5:1)19 ± 3.10.89 ± 0.0621 ± 2.20.92 ± 0.08PKCδ + PKCε:PKCβI + PKCβII (5:1)20 ± 1.60.91 ± 0.0719 ± 2.00.48 ± 0.06PKCδ + PKCζ:PKCβI + PKCβII (5:1)21 ± 1.40.88 ± 0.0721 ± 1.80.45 ± 0.07G, single channel conductance; Po, the open probability of the channel; *, in the presence of 0.983 μm[Ca2+]free; **, in the presence of 2.24 μm [Ca2+]free. Bilayers were bathed with symmetrical 100 mm NaCl, 10 mm MOPS-Tris, 100 μm EGTA, 50 nm[Ca2+]free, pH 7.4. Phosphorylation mixture contained 10 ng/ml PKC or its isoforms, 5 μm diacyl glycerol, 100 μm Mg-ATP. At higher Ca2+ concentrations (0.983 and 2.24 μm), PKCβI and PKCβII also inhibited BNaC2 channel activity, and this inhibition was in addition to the inhibitory effect of Ca2+ itself on the channel. Open table in a new tab G, single channel conductance; Po, the open probability of the channel; *, in the presence of 0.983 μm[Ca2+]free; **, in the presence of 2.24 μm [Ca2+]free. Bilayers were bathed with symmetrical 100 mm NaCl, 10 mm MOPS-Tris, 100 μm EGTA, 50 nm[Ca2+]free, pH 7.4. Phosphorylation mixture contained 10 ng/ml PKC or its isoforms, 5 μm diacyl glycerol, 100 μm Mg-ATP. At higher Ca2+ concentrations (0.983 and 2.24 μm), PKCβI and PKCβII also inhibited BNaC2 channel activity, and this inhibition was in addition to the inhibitory effect of Ca2+ itself on the channel. Because of the functional effects of PKC and its isoforms on BNaC2 activity in planar bilayers, we hypothesized that BNaC2 should be a substrate for phosphorylation by this kinase and its isoforms. As illustrated in Fig.3 A, BNaC2 can indeed be specifically phosphorylated by PKC (lanes 1 and2). Elimination of BNaC2 or inclusion of a PKC peptide inhibitor to the reaction mixture prevented phosphorylation of BNaC2 (lanes 3 and 4, respectively). Similarly, PKC isoforms (βI, βII, δ, ε, and ζ) phosphorylated BNaC2 (Fig.3 B). Elimination of BNaC2 protein from the reaction mixture prevented BNaC2 phosphorylation (data not shown). Whole-cell patch clamp experiments were performed on cultured human U87-MG glioma cells to test the hypothesis that PKCβI + PKCβII can inhibit the constitutively activated inward Na+ currents seen in these cells. As a prelude, Fig.4 presents representative whole-cell patch clamp records from a U87-MG cell before and after treatment with amiloride. Amiloride effectively blocked inward currents. Fig.5 represents the patch clamp results of the PKC experiments. Inclusion of 5 ng/ml PKCβII in the pipette solution had no effect on the inward currents (middle panel). In contrast, PKCβI + PKCβII abolished the inward currents (right panel). As an additional control, PKCζ also was without effect (data not shown), consistent with the bilayer findings (Table II).Figure 5Whole-cell patch clamp recordings from representative U87-MG cells. PKC isoforms were included in the pipette solution at a final concentration of 5 ng/ml. The chord conductance measured between −80 mV, and the reversal potential was (in pS) 5250 ± 1700 (basal), 7318 ± 2316 (PKCβII), and 2916 ± 987 (PKCβI + βII); n = 4 for each. The mean conductance value for the PKCβI + βII group was significantly different from the PKCβI (p < 0.005), the basal (p < 0.01), or the PKC-ζ (5350 ± 1891 pS; p < 0.01, not shown) groups.View Large Image Figure ViewerDownload (PPT) The effects of PKC phosphorylation on ion channel function can either be stimulatory or inhibitory depending upon the type of ion channel and cell type (46Ismailov I.I. Benos D.J. Kidney Int. 1995; 48: 1167-1179Abstract Full Text PDF PubMed Scopus (64) Google Scholar, 47Numann R. Catterall W.A. Scheuer T. Science. 1991; 254: 115-118Crossref PubMed Scopus (266) Google Scholar, 48Wang W. Sackin H. Giebisch G. Annu. Rev. Physiol. 1992; 54: 81-96Crossref PubMed Scopus (128) Google Scholar, 49Lo C.F. Numann R. Ann. N. Y. Acad. Sci. 1999; 868: 431-433Crossref PubMed Scopus (6) Google Scholar, 50Stea A. Soong T.W. Snutch T.P. Neuron. 1995; 15: 929-940Abstract Full Text PDF PubMed Scopus (194) Google Scholar, 51Yang J. Tsien R.W. Neuron. 1993; 10: 127-136Abstract Full Text PDF PubMed Scopus (152) Google Scholar). The effects of PKC on amiloride-sensitive Na+ transporting pathways were even more complex and diverse. Activation of PKC greatly diminishe" @default.
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• W2034930415 date "2002-11-01" @default.
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• W2034930415 title "Protein Kinase C Isoform Antagonism Controls BNaC2 (ASIC1) Function" @default.
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