Chloride and potassium conductances of mouse pancreatic zymogen granules are inversely regulated by a approximately 80-kDa mdr1a gene product.
Cl- and cation conductances were characterized in zymogen granules (ZG) isolated from the pancreas of wild-type mice (+/+) or mice with a homozygous disruption of the multidrug resistance P-glycoprotein gene mdr1a (-/-). Cl- conductance of ZG was assayed in isotonic KCl buffer by measuring osmotic lysis, which was induced by maximal permeabilization of ZG membranes (ZGM) for K+ with valinomycin due to influx of K+ through the artificial pathway and of Cl- through endogenous channels. To measure cation conductances, ZG (pHi 6.0-6.5) were suspended in buffered isotonic monovalent cation acetate solutions (pH 7.0). The pH gradient was converted into an outside-directed H+ diffusion potential by maximally increasing H+ conductance of ZGM with carbonyl cyanide m-chlorophenylhydrazone. Osmotic lysis of ZG was induced by H+ diffusion potential-driven influx of monovalent cations through endogenous channels and nonionic diffusion of the counterion acetate. ZGM Cl- conductances were not different in (-/-) and (+/+) mice (2.6 +/- 0.3 h-1 versus 3.1 +/- 0.2 h-1 (relative rate constant)). The nonhydrolyzable ATP analog adenosine 5'-(beta,gamma-methylene)triphosphate (AMP-PCP) (0.5 mM) activated the Cl- conductance both in (+/+) and (-/-) mice. However, activation of Cl- conductance by AMP-PCP was reduced in (-/-) mice as compared with (+/+) mice (5.0 +/- 0.4 h-1 versus 7.6 +/- 0.7 h-1; p < 0. 005). In contrast, ZGM K+ conductance was increased in (-/-) mice as compared with (+/+) mice (14.2 +/- 2.0 h-1 versus 8.5 +/- 1.2 h-1; p < 0.03). In the presence of 0.5 mm AMP-PCP, which completely blocks K+ conductance but leaves a nonselective cation conductance unaffected, there was no difference between (-/-) and (+/+) mice (5.3 +/- 0.7 h-1 versus 3.2 +/- 0.5 h-1). In Western blots of ZGM from wild-type mice, a polyclonal MDR1 specific antibody labeled a protein band of approximately 80 kDa. In mdr1a-deficient mice, the intensity of this band was reduced to 39 +/- 7% of the wild-type signal. This indicates that a mdr1a gene product of approximately 80 kDa enhances the AMP-PCP-activated fraction of mouse ZGM Cl- conductance and reduces AMP-PCP-sensitive K+ conductance.
Thévenod
F
F
II Department of Physiology, Medical Faculty, University of Saarland, 66421 Homburg/Saar, Federal Republic of Germany.
Hildebrandt
J P
JP
Striessnig
J
J
de Jonge
H R
HR
Schulz
I
I
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000172:deficiency* / Q000235:genetics / Q000502:physiology*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001692:Biological Transport / Q000187:drug effects; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D018118:Chloride Channels / Q000502:physiology*; D002712:Chlorides / Q000378:metabolism*; D003594:Cytoplasmic Granules / Q000187:drug effects / Q000502:physiology*; D018432:Drug Resistance, Multiple / Q000235:genetics*; D006801:Humans; D007425:Intracellular Membranes / Q000187:drug effects / Q000502:physiology; D007700:Kinetics; D051379:Mice; D008817:Mice, Mutant Strains; D008954:Models, Biological; D008969:Molecular Sequence Data; D010179:Pancreas / Q000502:physiology*; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D011188:Potassium / Q000378:metabolism*; D015221:Potassium Channels / Q000502:physiology*
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000906:Antibodies; D018118:Chloride Channels; D002712:Chlorides; D010446:Peptide Fragments; D015221:Potassium Channels; C005147:5'-adenylyl (beta,gamma-methylene)diphosphonate; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D000255:Adenosine Triphosphate; C002630:alpha,beta-methyleneadenosine 5'-triphosphate; D011188:Potassium