Physical and functional coupling of electrogenic Na+/HCO3 cotransport and carbonic anhydrases in the myocardium

ORLOWSKI, A; DE GIUSTI, VC; ÁLVAREZ, BV; AIELLO, EA

San Francisco

Congreso; 54 meeting Biophysical Society; 2010

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 coimmunoprecipitation 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