Chloride and potassium conductances of mouse pancreatic zymogen granules are inversely regulated by a approximate to 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 (pH(i) 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 approximate to 80 kDa, In mdrla-deficient mice, the intensity of this band was reduced to 39 +/- 7% of the wild-type signal, This indicates that a mdrla gene product of approximate to 80 kDa enhances the AMP-PCP-activated fraction of mouse ZGM Cl- conductance and reduces AMP-PCP-sensitive K+ conductance.