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

Abstract

In rat pancreatic zymogen granules (ZG), an ATP-sensitive K⁺ conductance and a Cl⁻ conductance have been characterized that are inversely regulated by an ≈65-kDa multidrug resistance P-glycoprotein (mdr1) gene product. In search of a label for purification of this protein, we found that the dihydropyridine derivative (−)-[³H]BZDC-DHP, a recently developed high-affinity ligand for Mdr1, binds with similar affinity to ZG membranes (ZGM) (K _d = 6.2 nM). Binding was inhibited by nanomolar concentrations of the L-type Ca²⁺ channel blockers azidopine and verapamil and by micromolar concentrations of the K⁺ channel blockers glibenclamide and quinidine. Inhibition 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 inhibited the K⁺ conductance in ZGs, whereas the Cl⁻conductance was not affected. In photoaffinity labeling experiments, (−)-[³H]BZDC-DHP surprisingly specifically and selectively labeled a ≈19-kDa protein in ZGM with a pharmacological profile identical with the high-affinity binding site but did not label a 65-kDa protein. The 19-kDa protein was purified by ion exchange chromatography and SDS-polyacrylamide gel electrophoresis and sequenced. The sequence obtained corresponds to ZG-16p, a recently cloned ZG protein with no apparent homology to Mdr1. The identity of the 19-kDa protein was confirmed by immunoprecipitation of (−)-[³H]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.