Skip to main content
SpringerLink
Log in

GTP-binding proteins regulate high conductance anion channels in rat bile duct epithelial cells

  • Articles
  • Published:
The Journal of Membrane Biology Aims and scope Submit manuscript

Summary

Epithelial cells from the intrahepatic bile duct contribute to bile formation, but little is known of the cellular mechanisms responsible. In these studies, we have characterized the endogenous GTP-binding proteins (G proteins) present in these cells and evaluated their role in regulation of high conductance anion channels. G proteins were identified in purified plasma membranes of isolated bile duct epithelial cells using specific antisera on Western blots, and ion channel activity was measured in excised inside-out membrane patches using patch-clamp recording techniques. In patches without spontaneous channel activity, addition of cholera toxin to the cytoplasmic surface had no effect (n=10). Addition of pertussis toxin caused an activation of channels in 13/34 (38%) attempts, as detected by an increase in channel open probability. Activated channels were anion selective (gluconate/Cl− permeability ratio of 0.17±0.04) and had a unitary conductance of ∼380 pS. Channel open probability was also increased by the nonhydrolyzable GDP analogue guanosine 5′-0-(2-thiodiphosphate) in 8/14 (57%) attempts. In contrast, channel open probability was rapidly and reversibly decreased by the nonhydrolyzable analogue of GTP 5′guanylylimidodiphosphate in 7/9 (78%) attempts. Western blotting with specific antisera revealed that both Gi α−2 and Gi α−3 were present in significant amounts, whereas Gi α−1 and Go α were not detected. These studies indicate that in bile duct epithelial cells, high conductance anion channels are inhibited, in a membrane-delimited manner, by PTX-sensitive G proteins.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Alpini, G., Lenzi, R., Sarkozi, L., Tavoloni, N. 1988. Biliary physiology in rats with bile ductular hyperplasia. J. Clin Invest. 81:569–578

    Google Scholar 

  • Alpini, G., Lenzi, R., Zhai, W.R., Slott, P.A., Liu, M.H., Sarkozi, L., Tavoloni, N. 1989. Bile secretory function of intrahepatic biliary epithelium in the rat. Am. J. Physiol. 257:G124-G133

    Google Scholar 

  • Birnbaumer, L., Codina, J., Yatani, A., Mattera, R., Graf, R., Olate, J., Themmen, A.P.N., Liao, C.-F., Sanford, J., Okabe, K., Imoto, Y., Zhou, Z., Abramowitz, J., Suki, W.N., Hamm, H.E., Iyengar, R., Birnbaumer, M., Brown, A.M. 1989. Molecular basis of regulation of ionic channels by G proteins. Recent Prog. Horm. Res. 45:121–208

    Google Scholar 

  • Breitwieser, G.E. 1991. G protein-mediated ion channel activation. Hypertension 17:684–692

    Google Scholar 

  • Brown, A.M. 1990. Ionic channels and their regulation by G protein subunits. Annu. Rev. Physiol. 52:197–213

    Google Scholar 

  • Brown, A.M., Birnbaumer, L. 1988. Direct G protein gating of ion channels. Am. J. Physiol. 254:H401-H410

    Google Scholar 

  • Casey, P.J., Gilman, A.G. 1988. G-protein involvement in receptor-effector coupling. J. Biol. Chem. 263:2577–2580

    Google Scholar 

  • Cerbai, E., Klöckner, U., Isenberg, G. 1988. The α-subunit of the GTP binding protein activates muscarinic potassium channels of the atrium. Science 240:1782–1783

    Google Scholar 

  • Cohen-Armon, M., Garty, H., Sokolovsky, M. 1988. G-protein mediates voltage regulation of agonist binding to muscarinic receptors: effects on receptor-Na2+ channel interaction. Biochemistry 27:368–374

    Google Scholar 

  • Fargon, F., McNaughton, P.A., Sepúlveda, F.V. 1990. Possible involvement of GTP-binding proteins in the deactivation of an inwardly rectifying K2+ current in enterocytes isolated from guinea-pig small intestine. Pfluegers Arch. 417:240–242

    Google Scholar 

  • Farouk, M., Vigna, S.R., McVey, D.C., Meyers, W.C. 1992. Localization and characterization of secretin binding sites expressed by rat bile duct epithelium. Gastroenterology 102:963–968

    Google Scholar 

  • Goldsmith, P., Rossiter, K., Carter, A., Simonds, W., Unson, C.G., Vinitsky, R., Spiegel, A.M. 1988. Identification of the GTP-binding protein encoded by Gi3 complementary DNA. J. Biol. Chem. 263:6476–6479

    Google Scholar 

  • Green, N., Alexander, H., Olson, A., Alexander, S., Shinnick, T.M., Sutcliffe, J.G., Lerner, R.A. 1982. Immunogenic structure of the influenza virus hemagglutin. Cell 28:477–487

    Google Scholar 

  • Hamill, O.P., Marty, A., Neher, A., Sakmann, B., Sigworth, E.J. 1981. Improved patch-clamp techniques for high resolution recording from cells and cell-free patches. Pfluegers Arch. 391:85–100

    Google Scholar 

  • Inoue, M., Kuriyama, H. 1991. Muscarinic receptor is coupled with a cation channel through a GTP-binding protein in guinea-pig chromaffin cells. J. Physiol. 436:511–529

    Google Scholar 

  • Inoue, R., Isenberg, G. 1990. Acetylcholine activates nonselective cation channels in guinea pig ileum through a G protein. Am. J. Physiol. 258:C1173-C1178

    Google Scholar 

  • Ishii, M., Vroman, B., LaRusso, N.F. 1989. Isolation and morphological characterization of bile duct epithelial cells from normal rat liver. Gastroenterology 97:1236–1247

    Google Scholar 

  • Johnson, G.L., Kaslow, H.R., Bourne, H.R. 1978. Reconstitution of cholera toxin-activated adenylate cyclase. Proc. Natl. Acad. Sci. USA 75:3113–3117

    Google Scholar 

  • Katada, T., Ui, M. 1982. Direct modification of the membrane adenylate cyclase system by islet-activating protein due to ADP-ribosylation of a membrane protein. Proc. Natl. Acad. Sci. USA 79:3129–3133

    Google Scholar 

  • Kato, A., Gores, G.J., LaRusso, N.F. 1992. Secretin stimulates exocytosis in isolated bile duct epithelial cells by a cyclic AMP-mediated mechanism. J. Biol. Chem. 267:15523–15529

    Google Scholar 

  • Kirsch, G.E., Yatani, A., Codina, J., Birnbaumer, L., Brown, A.M. 1988. α-subunit of Gk activated atrial K2+ channels of chick, rat, and guinea pig. Am. J. Physiol. 254:H1200-H1205

    Google Scholar 

  • Light, D.B., Ausiello, D.A., Stanton, B.A. 1989. Guanine nucleotide-binding protein, α i−3, directly activates a cation channel in rat renal inner medullary collecting duct cells. J. Clin. Invest. 84: 352–356

    Google Scholar 

  • Light, D.B., Schwiebert, E.M., Fejes-Toth, G., Naray-Fejes-Toth, A., Karlson, K.H., McCann, F.V., Stanton, B.A. 1990. Chloride channels in the apical membrane of cortical collecting duct cells. Am. J. Physiol. 258:F273-F280

    Google Scholar 

  • Linder, M.E., Gilman, A.G. 1991. Purification of recombinant Gi-α and Go-α proteins from Escherichia coli. Methods Enzymol. 195:202–215

    Google Scholar 

  • Logothetis, D.E., Kim, D., Northup, J.K., Neer, E.J., Clapham, D.E. 1988. Specificity of action of guanine nucleotide-binding regulatory protein subunits on the cardiac muscarinic K2+ channel. Proc. Natl. Acad. Sci. USA 85:5814–5818

    Google Scholar 

  • Lynch, C.J., Blackmore, P.F., Johnson, E.H., Wange, R.L., Krone, P.K., Exton, J.H. 1989. Guanine nucleotide binding regulatory proteins and adenylate cyclase in livers of streptozotocin- and BB/Wor-diabetic rats. J. Clin. Invest. 83:2050–2062

    Google Scholar 

  • Mathis, G.A., Walls, S.A., Sirica, A.E. 1988. Biochemical characteristics of hyperplastic rat bile ductular epithelial cells cultured “on top” and “inside” different extracellular matrix substitutes. Cancer Res. 48:6145–6153

    Google Scholar 

  • McGill, J.M., Basavappa, S., Fitz, J.G. 1992. Characterization of high-conductance anion channels in rat bile duct epithelial cells. Am. J. Physiol. 262:G703-G710

    Google Scholar 

  • Moore, D.J., Moore, D.M. 1989. Preparation of mammalian plasma membranes by aqueous two-phase partition. Biotechniques 7:946–958

    Google Scholar 

  • Mumby, S., Pang, I.-K., Gilman, A.G., Sternweiss, P.C. 1988. Chromatographic resolution and immunologic identification of the α−40 and α−41 subunits of guanine nucleotide-binding regulatory proteins. J. Biol. Chem. 263:2020–2026

    Google Scholar 

  • Mumby, S.M., Gilman, A.G. 1991. Synthetic peptide antisera with determined specificity for G protein α or β subunits. Methods Enzymol. 195:215–233

    Google Scholar 

  • Nathanson, M.H., Boyer, J.L. 1991. Mechanisms and regulation of bile secretion. Hepatology 14:551–566

    Google Scholar 

  • Nelson, D.J., Tang, J.M., Palmer, L.G. 1984. Single channel recordings of apical membrane chloride conductance in A6 epithelial cells. J. Membrane Biol. 80:81–89

    Google Scholar 

  • Ross, E.M., Gilman, A.G. 1977. Resolution of some components of adenylate cyclase necessary for catalytic activity. J. Biol. Chem. 252:6966–6969

    Google Scholar 

  • Schlichter, L.C., Grygorczyk, R., Pahapill, P.A., Grygorczyk, C. 1990. A large, multiple-conductance chloride channel in normal human T lymphocytes. Pfluegers Arch. 416:413–421

    Google Scholar 

  • Schneider, G.T., Cook, D.I., Gage, P.W., Young, J.A. 1985. Voltage sensitive, high-conductance chloride channels in the luminal membrane of cultured pulmonary alveolar (type II) cells. Pfluegers Arch. 404:354–357

    Google Scholar 

  • Schwiebert, E.M., Light, D.B., Fejes-Toth, G., Naray-Fejes-Toth, A., Stanton, B. 1990. A GTP-binding protein activates chloride channels in a renal epithelium. J. Biol. Chem. 265:7725–7728

    Google Scholar 

  • Sirica, A.E., Sattler, C.A., Cihla, H.P. 1985. Characterization of a primary bile ductular cell culture from the livers of rats during extrahepatic cholestasis. Am J. Pathol. 120:67–78

    Google Scholar 

  • Sirica, A.E., Mathis, G.A., Sano, N., Elmore, L.W. 1990. Isolation, culture, and transplantation of intrahepatic biliary epithelial cells and oval cells. Pathobiology 58:44–64

    Google Scholar 

  • Sirica, A.E., Cihla, H.P. 1984. Isolation and partial characterization of oval and hyperplastic bile ductular cell-enriched populations from the livers of carcinogen and noncarcinogen-treated rats. Cancer Res. 44:3454–3466

    Google Scholar 

  • Slott, P.A., Liu, M.H., Tavoloni, N. 1990. Origin, pattern, and mechanism of bile duct proliferation following biliary obstruction in the rat. Gastroenterology 99:466–477

    Google Scholar 

  • Sunyer, T., Monastirsky, B., Codina, J., Birnbaumer, L. 1989. Studies on nucleotide and receptor regulation of Gi proteins: Effects of pertussis toxin. Mol. Endocrinol. 3:1115–1124

    Google Scholar 

  • Tilly, B.C., Kansen, M., van Gageldonk, P.G.M., van den Berghe, N., Galjaard, H., Bijman, J., de Jonge, H.R. 1991. G-proteins mediate intestinal chloride channel activation. J. Biol. Chem. 266:2036–2040

    Google Scholar 

  • Uhing, R.J., Polakis, P.G., Snyderman, R. 1987. Isolation of GTP-binding proteins from myeloid HL-60 cells. J. Biol. Chem. 262:15575–15579

    Google Scholar 

  • Yatani, A., Codina, J., Imoto, Y., Reeves, J.P., Birnbaumer, L., Brown, A.M. 1987. A G protein directly regulates mammalian cardiac calcium channels. Science 238:1288–1292

    Google Scholar 

  • Yatani, A., Mattera, R., Codina, J., Graf, R., Okabe, K., Padrell, E., Iyengar, R., Brown, A.M., Birnbaumer, L. 1988. The G protein-gated atrial K2+ channel is stimulated by three distinct Gi α-subunits. Nature 336:680–682

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Duke University Medical Center, Division of Gastroenterology, 27710, Durham, North Carolina, USA

    James M. McGill, Srisaila Basavappa & J. Gregory Fitz

  2. Departments of Medicine and Cell Biology, Division of Gastroenterology, 27710, Durham, North Carolina, USA

    Thomas W. Gettys

Authors
  1. James M. McGill
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Thomas W. Gettys
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Srisaila Basavappa
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. J. Gregory Fitz
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

We gratefully acknowledge the assistance of Marwan Farouk, M.D. in the preparation of bile duct epithelial cells, Lucy Seger in the identification of the G proteins, C.F. Starmer in channel analysis, and P.J. Casey for the gift of bacteria expressing the different G-protein α-subunits. This work was supported in part by grants from the National Institutes of Health DK43278 (to J.G.F.), DK42486 (to T.W.G.), and DK07568 (to J.M.M.); American Gastroenterological Association/G.D. Searle Research Scholar Award (to J.G.F.) and an American Gastroenterological Association Advanced Research Training Award (J.M.M.).

Rights and permissions

Reprints and permissions

About this article

Cite this article

McGill, J.M., Gettys, T.W., Basavappa, S. et al. GTP-binding proteins regulate high conductance anion channels in rat bile duct epithelial cells. J. Membarin Biol. 133, 253–261 (1993). https://doi.org/10.1007/BF00232024

Download citation

  • Received:

  • Revised:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00232024

Key Words

Search

Navigation