MONOCLONAL-ANTIBODIES AGAINST MDR1 P-GLYCOPROTEIN INHIBIT CHLORIDE CONDUCTANCE AND LABEL A 65KDA PROTEIN IN PANCREATIC ZYMOGEN GRANULE MEMBRANES

The regulation of Cl- and cation conductances by the nonhydrolyzabIe A TP analog adenosine 5'-(beta,gamma-methylene)triphosphate (AMP-PCP) wa s characterized in isolated zymogen granules (ZG) from pancreatic acin ar cells. ZG were purified from rat pancreas homogenate by Percoll gra dient centrifugation. Cl- conductance was assayed by suspending ZG in isotonic KCl buffer and measuring osmotic lysis induced by maximal per meabilization of ZG membranes (ZGM) for K+ with the K+ ionophore valin omycin (Val). This resulted in influx of K+ through the artificial pat hway and of Cl- through endogenous channels. To measure cation conduct ances ZG (pH(i) approximate to 6) were suspended in pH 7 buffered isot onic monovalent cation acetate salts. The pH gradient was converted in to an outside-directed H+ diffusion potential by maximally increasing H+ conductance of ZGM with the protonophore carbonyl cyanide p-chlorop henylhydrazone. Osmotic lysis of ZG was induced by H+ diffusion potent ial driven influx of monovalent cations through endogenous channels an d non-ionic diffusion of the counterion acetate. In the absence of Val , ZG were stable in KCl buffer up to 2 h. AMP-PCP enhanced osmotic lys is approximate to 4-fold compared to control, due to activation of Cl- conductance by AMP-PCP and K+ influx through an AMP-PCP-insensitive n onselective cation pathway, which could be blocked by 0.1 mM Ba2+, 0.5 mM quinine, or 0.2 mM flufenamate. In addition, a K+ and Rb+ selectiv e cation conductance was found which was completely blocked by 0.5 mM AMP-PCP or 0.5 mM quinine. AMP-PCP induced C1(-) conductance was stron gly inhibited by two monoclonal antibodies against MDR1 P-glycoprotein (JSB-1 and 0219; 5-10 mu g/ml), but not by a monoclonal antibody agai nst the cystic fibrosis transmembrane conductance regulator (M3A7; 5 m u g/ml) or by mouse IgG. The AMP-PCP insensitive nonselective cation c onductance was not blocked by monoclonal antibodies against MDR1 P-gly coprotein (MDR1). Immunoblot studies of ZG membranes revealed the pres ence of a major immunoreactive protein band of approximate to 65 kDa w ith both monoclonal antibodies against MDR1, but no protein of the app roximate size of MDR1 (approximate to 170 kDa) was detected. We propos e that the C1(-) channel or a regulator of the channel, that is activa ted by the non-hydrolyzable ATP analog AMP-PCP in ZG membranes, is a m ember of the ATP binding cassette superfamily of transporters and may have homology to MDR1 P-glycoprotein.