CL--HCO3- EXCHANGE IN DEVELOPING NEONATAL RAT CARDIAC-CELLS - BIOCHEMICAL-IDENTIFICATION AND IMMUNOLOCALIZATION OF BAND 3-LIKE PROTEINS

The Cl--HCO3- exchanger is the main anionic exchanger (AE) that allevi ates alkaline loads in cardiac cells. We recently identified in adult ventricular cells two membrane proteins (80 and 120 kD) immunologicall y related to the erythroid band 3 and likely to mediate the anion exch ange. In the present study, we further investigated the Cl--HCO3- exch anger activity concomitantly with the expression and intracellular loc alization of the band 3-like proteins during the development of neonat al rat cardiac cells maintained in culture for 17 days. Microspectrofl uorometric measurements of pH, in single cells show that neonatal rat cardiomyocytes display a fully functional DIDS-sensitive Cl--HCO3- exc hanger at early stages of development. Neither basal pH(i) nor the ani on exchange activity changes with different stages of the culture. In Western blotting with an anti-whole erythroid band 3 antibody, we foun d both the 80- and the 120-kD band 3-like proteins in whole heart and cultured neonatal cardiac cells. The 80-kD protein was also recognized by an anti-AE1 antiserum, whereas the 120-kD protein was specifically detected by an anti-cardiac AE3 antibody. Thus, we propose that the p roteins are encoded by two different genes, AE1 and AE3, respectively. Subcellular fractionation of isolated and cultured cardiomyocytes rev ealed the presence of both proteins in the membrane, nuclear, and myof ibril fractions. The results obtained in biochemical experiments corro borate the confocal images of immunostained neonatal cells, which demo nstrate perinuclear location of band 3-like proteins at an early stage of development and their appearance within myofilaments after cell ma turation. Colocalization of band 3-like proteins with specific markers of the Golgi apparatus, wheat germ agglutinin and CTR433 antibody, su ggests the presence of these proteins in the Golgi area. The later dec oration of repetitive striations between each sarcomere indicates that the band 3-like proteins are assigned in development within a compart ment similar to costameres, areas of attachment of myofibrils to sarco lemma. These areas are likely to play a major role in signal transduct ion of neurohormonal stimuli.