Binding of carbonic anhydrase IX to extracellular loop 4 of the NBCe1 Na+/HCO3− cotransporter enhances NBCe1-mediated HCO3− influx in the rat heart

Na+/HCO3− cotransporter (NBC)e1 catalyze the electrogenic movement of 1 Na+:2 HCO3− into cardiomyocytes cytosol. NBC proteins associate with carbonic anhydrases (CA), CAII, and CAIV, forming a HCO3− transport metabolon. Herein, we examined the physical/functional interaction of NBCe1 and transmembrane CAIX in cardiac muscle. NBCe1 and CAIX physical association was examined by coimmunoprecipitation, using rat ventricular lysates. NBCe1 coimmunoprecipitated with anti-CAIX antibody, indicating NBCe1 and CAIX interaction in the myocardium. Glutathione-S-transferase (GST) pull-down assays with predicted extracellular loops (EC) of NBCe1 revealed that NBCe1-EC4 mediated interaction with CAIX. Functional NBCe1/CAIX interaction was examined using fluorescence measurements of BCECF in rat cardiomyocytes to monitor cytosolic pH. NBCe1 transport activity was evaluated after membrane depolarization with high extracellular K+ in the presence or absence of the CA inhibitors, benzolamide (BZ; 100 μM) or 6-ethoxyzolamide...