Relationship between a HCO3- -permeable conductance and a CLCA protein from rat pancreatic zymogen granules

Ca(2+)-induced enzyme secretion in the exocrine pancreas is not completely understood. We have proposed that Ca(2+)-induced enzyme secretion in the exocrine pancreas involves activation of ion conductances in the membrane of zymogen granules (ZG). Here we have identified a Ca(2+)-activated anion conductance in rat pancreatic ZG membranes (ZGM). Ca(2+) (2.5-50 microM) increased the conductance for I(-), NO(3)(-), Br(-), or HCO(3)(-), but not for Cl(-), as determined by the rate of valinomycin-induced osmotic lysis of ZG suspended in isotonic K(+)-salts. 4,4'-Diisothiocyanatodihydrostilbene-2,2'-disulfonate (100 microM) or 25 microM dithiothreitol strongly inhibited Ca(2+)-dependent lysis. The permeability sequence, Ca(2+) dependence, and inhibitor sensitivity of ZG anion conductance are reminiscent of a family of epithelial Ca(2+)-activated anion channels (CLCA). CLCA expression was confirmed by RT-PCR with rat pancreatic mRNA and mouse CLCA1 primers. A PCR product (580bp) exhibited 81%, 77%, and 57% amino acid similarity to the three mouse isoforms mCLCA-1, -2, and -3 (mgob-5), respectively. Antibodies against bovine tracheal CLCA1 showed CLCA expression in ZGM by immunoblotting, immunoperoxidase light microscopy, and immunogold labeling. These findings suggest that a CLCA-related protein could account for the Ca(2+)-activated HCO(3)(-) conductance of rat pancreatic ZGM and contribute to hormone-stimulated enzyme secretion.