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.
Footnotes
-
Send reprint requests to: Frank Thévenod, M.D., Ph.D., II. Department of Physiology, Medical Faculty, University of Saarland, 66421 Homburg/Saar, Germany. E-mail:frank.thevenod{at}med-rz.uni-sb.de
-
This study was supported by the Deutsche Forschungsgemeinschaft (SFB 246-C6 and Th 345/6-1 to F.T.).
- Abbreviations:
- ZG
- zymogen granule
- ZGM
- ZG membrane
- Mdr1
- multidrug resistance P-glycoprotein
- IEC
- ion exchange chromatography
- DIDS
- 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid
- AMP-PCP
- adenosine 5′-(β,γ-methylenetriphosphate)
- PAGE
- polyacrylamide gel electrophoresis
- BZDC-DHR
- 2,6-dimethyl-4-(2-(trifluoromethyl)-phenyl)-1,4-dihydropyridine-3,5-dicarboxylic acid {2-[3-(4-benzoylphenyl)propionylamino]ethyl}ester ethyl ester
- Received June 1, 1999.
- Accepted October 28, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
MolPharm articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|