Photoaffinity labeling and purification of ZG-16p, a high-affinity dihydropyridine binding protein of rat pancreatic zymogen granule membranes that regulates a K+-selective conductance

In rat pancreatic zymogen granules (ZG), an ATP-sensitive K+ conductance an d a Cl- conductance have been characterized that are inversely regulated by an approximate to 65-kDa multidrug resistance P-glycoprotein (mdr1) gene p roduct. In search of a label for purification of this protein, we found tha t the dihydropyridine derivative (2)-[H-3]BZDC-DHP, a recently developed hi gh-affinity ligand for Mdr1, binds with similar affinity to ZG membranes (Z GM) (K-d = 6.2 nM). Binding was inhibited by nanomolar concentrations of th e L-type Ca2+ channel blockers azidopine and verapamil and by micromolar co ncentrations of the K+ channel blockers glibenclamide and quinidine. Inhibi tion by glibenclamide was noncompetitive. The Mdr1 modulators cyclosporin A and vinblastine did not inhibit binding, which is different from Mdr1. In addition, only (+/-)-BZDC-DHP, azidopine, and verapamil selectively inhibit ed the K+ conductance in ZGs, whereas the Cl- conductance was not affected. In photoaffinity labeling experiments, (2)-[H-3]BZDC-DHP surprisingly spec ifically and selectively labeled a approximate to 19-kDa protein in ZGM wit h a pharmacological profile identical with the high-affinity binding site b ut did not label a 65-kDa protein. The 19-kDa protein was purified by ion e xchange chromatography and SDS-polyacrylamide gel electrophoresis and seque nced. The sequence obtained corresponds to ZG-16p, a recently cloned ZG pro tein with no apparent homology to Mdr1. The identity of the 19-kDa protein was confirmed by immunoprecipitation of (2)-[H-3]BZDC-DHP-labeled ZGM with an anti-ZG-16p antibody. Furthermore, it is shown that ZG-16p is associated with the ZGM. We propose that ZG-16p, as part of the submembranous granule matrix, regulates the ATP-sensitive K+ conductance of ZGs.