© 1995 American Heart Association, Inc.
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Cl--HCO3- Exchange in Developing Neonatal Rat Cardiac Cells
Biochemical Identification and Immunolocalization of Band 3–Like Proteins
From the Laboratoire Physiopathologie Cardiovasculaire (I.K., M.P., G.V.), INSERM U-390, Hôpital Arnaud de Villeneuve, Montpellier, France, and Institut Jacques Monod (R.C.), CNRS UMR 9922, Universite Paris-7.
Correspondence to Dr Irina Korichneva, Laboratoire Physiopathologie Cardiovasculaire, INSERM U-390, Hôpital Arnaud de Villeneuve, 371, Avenue du Doyen Gaston Giraud, 34295 Montpellier Cedex 5, France.
Abstract The Cl--HCO3- exchanger is the main anionic exchanger (AE) that alleviates alkaline loads in cardiac cells. We recently identified in adult ventricular cells two membrane proteins (80 and 120 kD) immunologically related to the erythroid band 3 and likely to mediate the anion exchange. In the present study, we further investigated the Cl--HCO3- exchanger activity concomitantly with the expression and intracellular localization of the band 3–like proteins during the development of neonatal rat cardiac cells maintained in culture for 17 days. Microspectrofluorometric measurements of pHi in single cells show that neonatal rat cardiomyocytes display a fully functional DIDS-sensitive Cl--HCO3- exchanger at early stages of development. Neither basal pHi nor the anion exchange activity changes with different stages of the culture. In Western blotting with an anti–whole erythroid band 3 antibody, we found 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 proteins are encoded by two different genes, AE1 and AE3, respectively. Subcellular fractionation of isolated and cultured cardiomyocytes revealed the presence of both proteins in the membrane, nuclear, and myofibril fractions. The results obtained in biochemical experiments corroborate the confocal images of immunostained neonatal cells, which demonstrate perinuclear location of band 3–like proteins at an early stage of development and their appearance within myofilaments after cell maturation. Colocalization of band 3–like proteins with specific markers of the Golgi apparatus, wheat germ agglutinin and CTR433 antibody, suggests the presence of these proteins in the Golgi area. The later decoration of repetitive striations between each sarcomere indicates that the band 3–like proteins are assigned in development within a compartment similar to costameres, areas of attachment of myofibrils to sarcolemma. These areas are likely to play a major role in signal transduction of neurohormonal stimuli.
Key Words: pH regulation • costamere • Golgi complex
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