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 and a Cl(-) conductance have been characterized that are inversely regulated by an approximately 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 (-)-[(3)H]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 Ca(2+) 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, (-)-[(3)H]BZDC-DHP surprisingly specifically and selectively labeled a approximately 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 (-)-[(3)H]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.
Braun
M
M
II. Department of Physiology, Medical Faculty, University of Saarland, Homburg/Saar, Germany.
Thévenod
F
F
D000255:Adenosine Triphosphate / Q000378:metabolism; D000345:Affinity Labels; D000818:Animals; D001577:Benzophenones / Q000494:pharmacology; D001665:Binding Sites; D004095:Dihydropyridines / Q000378:metabolism / Q000494:pharmacology; D004792:Enzyme Precursors / Q000302:isolation & purification* / Q000378:metabolism; D066298:In Vitro Techniques; D037102:Lectins / Q000302:isolation & purification* / Q000378:metabolism; D008297:Male; D010179:Pancreas / Q000201:enzymology / Q000378:metabolism*; D017319:Photosensitizing Agents / Q000494:pharmacology; D026902:Potassium Channel Blockers; D051381:Rats; D017208:Rats, Wistar
C099532:2,6-dimethyl-4-(2-(trifluoromethyl)phenyl)-1,4-dihydropyridine-3,5-dicarboxylic acid (2-(3-(4-benzoylphenyl)propionylamino)ethyl) ester ethyl ester; D000345:Affinity Labels; D001577:Benzophenones; D004095:Dihydropyridines; D004792:Enzyme Precursors; D037102:Lectins; D017319:Photosensitizing Agents; D026902:Potassium Channel Blockers; C119740:ZG16 protein, rat; C038806:1,4-dihydropyridine; D000255:Adenosine Triphosphate