Summary
Differentiated neuroblastoma cells exhibit both the delayed rectifier potassium current (I K) and the M-current (I M). The present study was designed to determine the roles of protein kinase C (PKC) and of the calmodulin-binding protein 80K/MARCKS, a prominent substrate for PKC and possible regulator of these currents. Neuroblastoma x glioma (NG108-15) hybrid cells transfected with m1 muscarinic receptors were grown with 1% fetal bovine serum (FBS) without the prostaglandin E1 (PGE1) and isobutylmethylxanthine (IBMX) usually added in preparation for electrophysiological studies. Under these conditions, the usual pleomorphism was largely abolished, leaving two populations of small cells with stellate and spherically symmetrical geometries. Whole-cell patch clamping indicated that the two cell types had identical electrophysiological properties, displaying: I k, a small current through a “T-like” Ca2+ channel, and no M-current.
Stimulation with carbachol shifted the distribution of cells to a more stellate morphology within 24 hr and later (after 48 hr) reduced the PKC substrate 80K/MARCKS by 22±7%. In contrast to the stimulation of I k observed with cardiac cells, PKC activation produced only a small inhibition of I k, which was independent of carbachol pretreatment. Thus, PKC and 80K/MARCKS can be dissociated from the regulation of I k in neuroblastoma cells.
Supported in part by research grants from the National Institutes of Health (DK-40145 and EY-08343) and from the U.K. Medical Research Council.
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References
Bell, R.M. 1986. Protein kinase C activation by diacylglycerol second messengers. Cell 45:631–632
Brooks, S.F., Herget, T., Broad, S., Rozengurt, E. 1992. The expression of 80K/MARCKS, a major substrate of PKC, is down-regulated through both PKC-dependent and -independent pathways: Effects of bombesin, PDGF and cAMP. J. Biol. Chem. 267:14212–14218
Brooks, S.F., Herget, T., Erusalimsky, J.D., Rozengurt, E. 1991. Protein kinase C activation potently down-regulates the expression of its major substrate, 80K, in Swiss 3T3 cells. EMBO J. 10:2497–2505
Brown, D.A. 1988. M-currents: an update. TINS 11:294–299
Brown, D.A., Higashida, H. 1988a. Voltage- and calciumactivated potassium currents in mouse neuroblastoma x rat glioma hybrid cells. J. Physiol. 397:149–165
Brown, D.A., Higashida, H. 1988b. Inositol 1,4,5-triphosphate and diacylglycerol mimic bradykinin effects on mouse neuroblastoma x rat glioma hybrid cells. J. Physiol. 397:185–207
Buisson, B., Bottari, S.P., de Gasparo, M., Gallo-Payet, N., Payet, M.D. 1992. The angiotensin AT2 receptor modulates T-type calcium current in non-differentiated NG108–15 cells. FEBS Lett. 309:161–164
Cantiello, H.F., Stow, J.L., Prat, A.G., Ausiello, D.A. 1991. Actin filaments regulate epithelial Na+ channel activity. Am. J. Physiol. 261:C882-C888
Civan, M.M., Oler, A., Peterson-Yantorno, K., George, K., O'Brien, T.G. 1991. A Ca2+-independent form of protein kinase C may regulate Na+ transport across frog skin. J. Membrane Biol. 121:37–50
Cohen, P. 1989. The structure and regulation of protein phosphatases. Annu. Rev. Biochem. 58:453–508
Docherty, R.J., Robbins, J., Brown, D.A. 1991. NG 108–15 neuroblastoma x glioma hybrid cell line as a model neuronal system. In: Cellular Neurobiology: A Practical Approach. J. Chad and H. Wheal, editors. pp. 75–95. Oxford University, Oxford
Dubois, J.-M., Rouzaire-Dubois, B. 1991. Interaction of 4-aminopyridine with normal and chloramine-T-modified K channels of neuroblastoma cells. Pfluegers Arch. 419:93–100
Erusalimsky, J.D., Brooks, S.F., Herget, T., Morris, C., Rozengurt, E. 1991. Molecular cloning and characterization of the acidic 80-kDa protein kinase C substrate from rat brain. J. Biol. Chem. 266:7073–7080
Finkel, A.S., Redman, S. 1984. Theory and operation of a single microelectrode voltage clamp. J. Neurosci. Meth. 11:101–127
Fukuda, K., Higashida, H., Kubo, T., Maeda, A., Akiba, I., Bujo, H., Mishina, M., Numa, S. 1988. Selective coupling with K+ currents of muscarinic acetylcholine receptor subtypes in NG 108–15 cells. Nature 335:355–358
Graff, J.M., Gordon, J.I., Blackshear, P.J. 1989a. Myristoylated and nonmyristoylated forms of a protein are phosphorylated by protein kinase C. Science 246:503–506
Graff, J.M., Young, T.N., Johnson, J.D., Blackshear, P.J. 19896b. Phosphorylation-regulated calmodulin binding to a prominent cellular substrate for protein kinase C. J. Biol. Chem. 264:21818–21823
Guadagno, S.N., Borner, C., Weinstein, I.B. 1992. Altered regulation of a major substrate of protein kinase C in rat 6 fibroblasts overproducing PKCβI. J. Biol. Chem. 267:2697–2707
Hartwig, J.H., Thelen, M., Rosen, A., Janmey, P.A., Nairn, A.C., Aderem, A. 1992. MARCKS is an actin filament crosslinking protein regulated by protein kinase C and calcium-calmodulin. Nature 356:618–622
Herget, T., Brooks, S.F., Broad, S., Rozengurt, E. 1992. Relationship between the major protein kinase C substrates 80K and MARCKS: Members of a gene family or equivalent genes in different species. Eur. J. Biochem. 209:7–14
Hille, B. 1984. Ionic Channels of Excitable Membranes. Sinauer Associates, Sunderland, MA
Kiley, S.C., Parker, P.J., Fabbro, D., Jaken, S. 1992. Hormone- and phorbol ester-activated protein kinase C isozymes mediate a reorganization of the actin cytoskeleton associated with prolactin secretion in GH4C1 cells. Mol. Endocrinol. 6:120–131
Lester, D.S., Asher, C., Garty, H. 1988. Characterization of cAMP-induced activation of epithelial sodium channels. Am. J. Physiol. 254:C802-C808
Mangels, L.A., Gnegy, M.E. 1992. Carbachol stimulates binding of a photoreactive calmodulin derivative to calmodulinbinding proteins in intact SK-N-SH human neuroblastoma cells. J. Biol. Chem. 267:5847–5854
McIlroy, B.K., Walters, J.D., Blackshear, P.J., Johnson, J.D. 1991. Phosphorylation-dependent binding of a synthetic MARCKS peptide to calmodulin. J. Biol. Chem. 266:4959–4964
Nishizawa, Y. 1986. Studies and perspectives of protein kinase C. Science 233:305–312
Numann, R., Catterall, W.A., Scheuer, T. 1991. Functional modulation of brain sodium channels by protein kinase C phosphorylation. Science 254:115–118
Reeve, H.L., Peers, C. 1992. Blockade of delayed rectifier K+ currents in neuroblastoma x glioma hybrid (NG 108–15) cells by clofilium, a class III antidysrhythmic agent. Br. J. Pharmacol. 105:458–462
Robbins, J., Sim, J. A. 1990. A transient outward current in NG108–15 neuroblastoma x glioma hybrid cells. Pfluegers Arch. 416:130–137
Robbins, J., Trouslard, J., Marsh, S.J., Brown, D.A. 1992. Kinetic and pharmacological properties of the M-current in rodent neuroblastoma x glioma hybrid cells. J. Physiol. 451:159–185
Rosen, A., Keenan, K.F., Thelen, M., Nairn, A.N., Aderem, A. 1990. Activation of protein kinase C results in the displacement of its myristoylated, alanine-rich substrate from punctate structures in macrophage filipodia. J. Exp. Biol. 172:1211–1215
Rozengurt, E. 1986. Early signals in the mitogenic response. Science 234:161–166
Rozengurt, E., Sinnett-Smith, J. 1988. Early signals underlying the induction of the c-fos and c-myc genes in quiescent fibroblasts: Studies with bombesin and other growth factors. Progr. Nucl. Acid Res. Mol. Biol. 35:261–295
Schäfer, S., Béhé, P., Meves, H. 1991. Inhibition of the M current in NG 108–15 neuroblastoma x glioma hybrid cells. Pfluegers Arch. 418:581–591
Seykora, J.T., Ravetch, J.V., Aderem, A. 1991. Cloning and molecular characterization of the murine macrophage “68-kDa” protein kinase C substrate and its regulation by bacterial lipopolysaccharide. Proc. Natl. Acad. Sci. USA 88:2505–2509
Stabel, S., Rodriguez-Pena, A., Young, S., Rozengurt, E., Parker, P.J. 1987. Quantitation of protein kinase C by immunoblot-expression in different cell lines and response to phorbol esters. J. Cell. Physiol. 130:111–117
Stumpo, D.J., Graff, J.M., Albert, K.A., Greengard, P., Blackshear, P.J. 1989. Molecular cloning, characterization, and expression of a cDNA encoding the “80-to 87-kDa” myristoylated alanine-rich C kinase substrate: A major cellular substrate for protein kinase C. Proc. Natl. Acad. Sci. USA 86:4012–4016
Tempel, B.L., Jan, Y.N., Jan, L.Y. 1988. Cloning of a probable potassium channel gene from mouse brain. Nature 332:837–839
Thelen, M., Rosen, A., Nairn, A.C., Aderem, A. 1991. Regulation by phosphorylation of reversible association of a myristoylated protein kinase C substrate with the plasma membrane. Nature 351:320–322
Walsh, K.B., Kass, R.S. 1988. Regulation of a heart potassium channel by protein kinase A and C. Science 242:67–69
West, J.W., Numann, R., Murphy, B.J., Scheuer, T., Catterall, W.A. 1991. A phosphorylation site in the Na+ channel required for modulation by protein kinase C. Science 254:866–868
Yokoyama, S., Imoto, K., Kawamura, T., Higashida, H., Iwabe, N., Miyata, T., Numa, S. 1989. Potassium channels from NG108–15 neuroblastoma-glioma hybrid cells: Primary structure and functional expression from cDNAs. FEBS Lett. 259:37–42
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We thank Dr. Peter J. Parker for his generous gift of PKC, and Yvonne Vallis for her skillful assistance with the cultures and harvesting of the NG108-15 transfected cells.
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Civan, M.M., Robbins, J., Broad, S. et al. Whole-cell recording of neuroblastoma x glioma cells during downregulation of a major substrate, 80K/MARCKS, of protein kinase C. J. Membarin Biol. 133, 51–59 (1993). https://doi.org/10.1007/BF00231877
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DOI: https://doi.org/10.1007/BF00231877