Physical and Functional Coupling of Electrogenic Na+/HCO3- Cotransport and Carbonic Anhydrases in the Myocardium

Abstract

The Na+/HCO3- cotransport (NBC) is an important sarcolemmal acid extruder in cardiac muscle. Functionally electroneutral (NBC3, 1 Na+:1 HCO3-) and electrogenic (NBC1 and NBC4, 1 Na+:2 HCO3-) forms of the transporter have been characterized in the heart. Mammalian cardiac muscle expresses membrane bound carbonic anhydrases (CA) IV (CAIV), IX (CAIX) and XIV (CAXIV), and cytosolic CAII. Association of CAII and CAIV, and NBC1, was previously demonstrated in the kidney and heterologous systems. NBC1 and CA physical and functional interaction was explored in the rat heart by co-immunoprecipitation and intracellular pH measurement (pHi) experiments, respectively. CAII, CAIV, CAIX, and CAXIV were immunoprecipitated with anti-NBC1 antibody, using rat ventricular lysates. Conversely, non immune serum and irrelevant anti-glial fibrilar acidic protein antibodies failed to co-immunoprecipitated CAs with NBC1. NBC1 activity was investigated in isolated rat cardiomyocytes, using intracellular fluorescent measurements of BCECF-AM, to monitor pHi. Cardiomyocyte membrane potential depolarazing pulses (MPDP) were applied by addition of 45 mM extracellular K+, to study NBC1 activity. After 10 minutes of MPDP a significant intracellular alkalinization was detected (0.17±0.03 pH units; n=6, P<0.05). The alkalinization was fully cancelled with specific anti-NBC1 functionally inhibitory antibodies (0.02±0.02 pH units; n=5), indicating activation of NBC1 isoform. Similarly, the NBC1-mediated increase of pHi (0.17±0.02 pH units; n=11, P<0.05), was inhibited with a poorly membrane-permeant CA inhibitor, benzolamide (100 μM, 0.09±0.02 pH units; n=6, P<0.05), and a potent membrane-permeant CA inhibitor, ethoxyzolamide (100 μM, 0.05±0.01 pH units; n=6, P<0.05), demonstrating a functional coupling between NBC1 cotransport and extracellular CAs, and NBC1and intracellular CAs in the cardiac muscle, respectively. We demonstrated that the NBC1 Na+/HCO3- cotransport is functionally and physically coupled to both plasma membrane-anchored CAs and cytosolic CAII, forming a HCO3- transport metabolon in the myocardium.