Protein Kinase C Isoform Antagonism Controls BNaC2 (ASIC1) Function

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 of PKC isoforms BNaC2 Also, of BNaC2 an expression of the PKC isoforms could for an activated amiloride-sensitive Na+ current 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). were PKC, PKC and PKC were other were and all were with and was human glioma cells and normal astrocytes a of the of and P. N. Biochem. PubMed Scopus Google Scholar). The of the was was to PKC mRNA with a mixture was of of and reverse and and was with a of for for PCR by of for for and for for a of This was by a of for to The in for reverse transcriptase reverse to of PKCβI and reverse to of PKCβI and in a new were by were in and no and other human were found for the we to we used for human PKC isoform and as in PCR with of the PCR were by and by The we for isoform the PKC isoform and not the other of was confirmed by and with as as by of and was the J. P. Res. 12: PubMed Scopus Google Scholar) on a and were the of at for were used as not 13Reeh P.W. Kress M. Curr. Opin. Pharmacol. 2001; 1: 45-51Crossref PubMed Scopus (140) Google Scholar). The used for Western for different PKC isoforms in tumor cell lines was to that B. A. Keeton D. Fuller C.M. Benos D.J. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus (52) Google Scholar). The were In by PKC and its isoforms was by the of protein in a mixture of and of PKC or its of in protein was as B. A. Keeton D. Fuller C.M. Benos D.J. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus (52) Google Scholar). The was for at and the phosphorylated were by and by PKC activity was in the presence of or PKC for and were used M.S. Benos D.J. Am. J. Physiol. PubMed Google Scholar). were as M.S. Benos D.J. Am. J. Physiol. PubMed Google Scholar, G. A. J.P. L. Biophys. J. 1994; Full Text PDF PubMed Scopus Google Scholar). were with bilayers of a in were with The was used to the level of Biochem. PubMed Scopus Google Scholar). mixture of PKC or its the of BNaC2 and its by at the of was added to the extracellular of the channel currents were a with a as Benos D.J. J. Physiol. 1997; PubMed Scopus Google Scholar). channel were on current low at an to a patch clamp were on human U87-MG glioma cells as (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 in the at a of specific for PKCα, PKCδ, PKCε, and PKCζ was on glioma cells was for of human of glioblastoma tumor and and cell of which were and a 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 for and were in all of the mRNA was expressed in all was in expression of and results that PKCα, and were expressed in all of the glioma cell lines and that PKCβI and PKCβII were not expressed at all used for the of PKCβ the of PKCβI and Western was also to protein expression of PKCβI, and PKCζ in the astrocytes and and U87-MG cells to the results in astrocytes expressed PKCβI, all cell expressed and the astrocytes and but not expressed BNaC2 bilayers a functional amiloride-sensitive Na+ channel with a low of being in an Mapstone T.B. Gillespie G.Y. J. Fuller C.M. Benos D.J. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). However, in the to and the to the effects of PKC and its isoforms on a of the mixture holoenzyme to the BNaC2 to any effect on channel We the that specific PKC isoforms could BNaC2 activity in different The for this of was the PKC is a family of with at with a expression and Trends Biochem. Sci. 1994; Full Text PDF PubMed Scopus Google Scholar). (35Xiao H. Goldthwait D.A. Mapstone T. J. Neurosurg. 1994; 81: 734-740Crossref PubMed Scopus (52) Google Scholar, M. J. 1999; Google Scholar) have that and PKCζ in many tumor cell PKCβ is or compared with normal human astrocytes amiloride-sensitive Na+ currents were in of and 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) with We by PKCβI and PKCβII effects on BNaC2 activity in bilayers. added PKCβI nor PKCβII had any effect on BNaC2 activity but their combination BNaC2 to no in were This of BNaC2 activity by the PKCβI and PKCβII combination was to that of whole results support the that PKCβI and PKCβII at in bilayers, for the inhibitory effect of PKC on BNaC2 of the reported of PKCε and PKCζ and the that these isoforms could have their effects on BNaC2 we explored the effect of PKCε and PKCζ on BNaC2 We found that PKCε and PKCζ added or in did not have any effect on BNaC2 activity This to the differential of PKC isoform expression in in 5-fold excess of PKCε and PKCζ to PKCβI and PKCβII was added to the of BNaC2. This abolished the otherwise inhibitory effect of the PKCβI and PKCβII combination on BNaC2 activity in bilayers We did not this effect with a of isoforms Also, a 5-fold excess of or its combination with PKCε or PKCζ BNaC2 activity nor with the inhibitory influence of the PKCβI and PKCβII combination on BNaC2 activity effect of by PKC and its isoforms on properties of BNaC2 in or PKC PKCβII PKCβII PKCβII PKCβII channel the of the in the presence of in the presence of were with mixture PKC or its and PKCβI and PKCβII also inhibited BNaC2 channel and this was in to the inhibitory effect of on the in a new channel the of the in the presence of in the presence of were with mixture PKC or its and PKCβI and PKCβII also inhibited BNaC2 channel and this was in to the inhibitory effect of on the of the functional effects of PKC and its isoforms on BNaC2 activity in bilayers, we that BNaC2 a for by this kinase and its in BNaC2 phosphorylated by PKC of BNaC2 or of a PKC to the mixture of BNaC2 and PKC isoforms and phosphorylated BNaC2 of BNaC2 protein the mixture BNaC2 not patch clamp were on human U87-MG glioma cells to the that PKCβI PKCβII inhibit the activated inward Na+ currents in these cells. a patch clamp a U87-MG cell and with inward the patch clamp results of the PKC of PKCβII in the had no effect on the inward currents In PKCβI PKCβII abolished the inward currents an PKCζ also was effect not consistent with the findings patch clamp U87-MG cells. PKC isoforms were in the at a of The and the was and for The for the PKCβI βII was different the PKCβI the or the not The effects of PKC on ion channel function or inhibitory the of ion channel and cell Benos D.J. Full Text PDF PubMed Scopus Google Scholar, R. T. Science. 1991; PubMed Scopus Google Scholar, H. G. Annu. Rev. Physiol. PubMed Scopus Google Scholar, R. Ann. N. Y. Acad. Sci. 1999; 868: PubMed Scopus Google Scholar, A. Neuron. Full Text PDF PubMed Scopus Google Scholar, J. Neuron. Full Text PDF PubMed Scopus Google Scholar). The effects of PKC on amiloride-sensitive Na+ were and of PKC Na+ in cells M. Am. J. Physiol. PubMed Google Scholar, Am. J. Physiol. Google Scholar) and in the cell M. D.A. Am. J. Physiol. PubMed Google Scholar). PKC also the activity of amiloride-sensitive channels Y. Smith Keeton D. Benos D.J. Am. J. Physiol. PubMed Google Scholar). to these PKC had no effect on Na+ in the Chen L. B.C. J.A. Am. J. Physiol. Google Scholar) and K. J. Biol. PubMed Scopus Google Scholar). may reported effects of PKC on Na+ currents Am. J. Physiol. 1998; PubMed Google Scholar) an was by of may also specific or effects of PKC on Na+ M.S. J. Physiol. 2000; PubMed Scopus Google Scholar). The inhibitory influence of PKC and its isoforms on BNaC2 activity 2) was of its effect on degenerin (DEG)/ENaC PKC inhibited channel activity in bilayers or heterologous expression in M.S. Fuller C.M. Benos D.J. J. Physiol. 1996; PubMed Scopus Google Scholar). Our findings the of a effect of PKC and its isoforms on BNaC2. This is also by the that BNaC2 was by PKC and its isoforms 3) and by findings of et al. R.A. R. C.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus Google Scholar) that β and but not the by Moreover, et al. J. B. P. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus (79) Google Scholar) that PKC of may differential regulation of PKC protein of β and but not The of of PKC and BNaC2 not the participation of other in this A. Garcı́a-Añoveros J. Corey D.P. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, A.M. J.A. Price M.P. Welsh M.J. Biochem. J. 2002; PubMed Scopus Google Scholar, N. E. L. Lazdunski M. Lingueglia E. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) an members of ASIC family and the which also with may to the for PKC regulation of of reported PKC isoform regulation of ion J.A. Chen L. N. D. M. Res. 1997; PubMed Scopus Google Scholar, K. D. M. Am. J. Physiol. Physiol. 2000; PubMed Google Scholar, Am. J. Physiol. Physiol. 2001; PubMed Google Scholar, C.M. M. J.B. Am. J. Physiol. Physiol. 2001; PubMed Google Scholar, Y. D. M. Am. J. Physiol. Physiol. 2001; PubMed Google Scholar), of BNaC2 by different PKC isoforms is of PKCβI and PKCβII were to BNaC2 their combination was to a functional inhibitory effect in bilayers. Moreover, this inhibitory effect was in the or the presence of an of PKCε, and PKCζ excess of a PKCε and PKCζ compared with PKCβI and PKCβII, abolished the inhibitory influence of the on BNaC2 Also, of PKCβI and PKCβII to the of U87-MG cells abolished inward Na+ results at that at isoforms and for to a functional PKCε and PKCζ individually phosphorylated BNaC2 in were to functional effects on BNaC2 in bilayers individually or in combination. to BNaC2 in was functionally by the otherwise inhibitory effects of PKCβI and The of molecular PKCε and PKCζ effects in BNaC2 by PKCβI and PKCβII is but is possible that by PKCε and PKCζ the BNaC2 protein in a such that the of PKCβI and PKCβII as a of the involvement of the or different the that the differential expression of PKC isoforms as of many of the effect of PKC an role in glioma cell glioma cells have been reported to to of PKC activity normal astrocytes M. J. 1999; Google Scholar). PKC such as C and A.M. A. F. J. 2000; PubMed Scopus Google Scholar) of glioma cell Moreover, of in U87-MG cells to cells T. F. M. J. Neurosci. 1999; PubMed Scopus Google Scholar); this effect may to M.J. J.H. T.B. Neurosci. Lett. 2000; PubMed Scopus (52) Google Scholar). of and PKCε had no effect on cell but PKCζ A.M. A. F. J. 2000; PubMed Scopus Google Scholar). In of in U87-MG cells the R. E. M. I. G. G. F. P.M. C. Res. 2001; 61: Google Scholar). of a in cells also inhibited N. M. J. 2001; Google Scholar). that many PKC effects on and these effects may to an amiloride-sensitive Na+ current in malignant brain tumor to BNaC2 (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). In patch clamp experiments, a combination of PKCβI and PKCβII in the inhibited the inward currents The presence of PKCβII or PKCζ in the had no effect on the inward The pattern of effects were in of any effect by an PKC but presence of inhibitory effect of PKCβI and PKCβII combination. Our results 1) that PKCα, and were expressed in most all of the glioma cell lines that were and that PKCβ was not expressed at we confirmed that PKCε and PKCζ in (35Xiao H. Goldthwait D.A. Mapstone T. J. Neurosurg. 1994; 81: 734-740Crossref PubMed Scopus (52) Google Scholar, M. J. 1999; Google Scholar) and show for the that not as compared with normal Our show that PKC inhibited BNaC2 activity in bilayers. Also, we found that an of specific PKC and in the of any inhibitory effects of the other PKC isoforms on BNaC2 expression of BNaC2, and for that other members of the ASIC family, is specific to glioma is that PKC of ASICs contribute to its activity in glioma cells. This effect may in such as and to these cells. We D. P. Corey and J. Garcı́a-Añoveros of and for the of