Carbonic anhydrases enhance activity of endogenous Na-H exchangers and not the electrogenic Na/HCO3cotransporter NBCe1-A, expressed inXenopusoocytes

Key points According to theHCO3-metabolon hypothesis, direct association of cytosolic carbonic anhydrases (CAs) with the electrogenic Na/HCO(3)cotransporter NBCe1-A speeds transport by regenerating/consumingHCO3-. The present work addresses published discrepancies as to whether cytosolic CAs stimulate NBCe1-A, heterologously expressed inXenopusoocytes. We confirm the essential elements of the previous experimental observations, taken as support for theHCO3-metabolon hypothesis. However, using our own experimental protocols or those of others, we find that NBCe1-A function is unaffected by cytosolic CAs. Previous conclusions that cytosolic CAs do stimulate NBCe1-A can be explained by an unanticipated stimulatory effect of the CAs on an endogenous Na-H exchanger. Theoretical analyses show that, although CAs could stimulate non-HCO3-transporters (e.g. Na-H exchangers) by accelerating CO2/HCO3--mediated buffering of acid-base equivalents, they could not appreciably affect transport rates of NBCe1 or other transporters carryingHCO3-,CO3=, orNaCO3-ion pairs. TheHCO3-metabolon hypothesis predicts that cytosolic carbonic anhydrase (CA) binds to NBCe1-A, promotesHCO3-replenishment/consumption, and enhances transport. Using a short step-duration current-voltage (I-V) protocol withXenopusoocytes expressing eGFP-tagged NBCe1-A, our group reported that neither injecting human CA II (hCA II) nor fusing hCA II to the NBCe1-A carboxy terminus affects background-subtracted NBCe1 slope conductance (G(NBC)), which is a direct measure of NBCe1-A activity. Others - using bovine CA (bCA), untagged NBCe1-A, and protocols keeping holding potential (V-h) far from NBCe1-A's reversal potential (E-rev) for prolonged periods - found that bCA increases total membrane current (Delta I-m), which apparently supports the metabolon hypothesis. We systematically investigated differences in the two protocols. In oocytes expressing untagged NBCe1-A, injected with bCA and clamped to -40 mV, CO2/HCO3-exposures markedly decreaseE(rev), producing large transient outward currents persisting for >10 min and rapid increases in [Na+](i). Although the CA inhibitor ethoxzolamide (EZA) reduces both Delta I(m)and d[Na+](i)/dt, it does not reduceG(NBC). In oocytes not expressing NBCe1-A, CO2/HCO3-triggers rapid increases in [Na+](i)that both hCA II and bCA enhance in concentration-dependent manners. These d[Na+](i)/dt increases are inhibited by EZA and blocked by EIPA, a Na-H exchanger (NHE) inhibitor. In oocytes expressing untagged NBCe1-A and injected with bCA, EIPA abolishes the EZA-dependent decreases in Delta I(m)and d[Na+](i)/dt. Thus, CAs/EZA produce their Delta I(m)and d[Na+](i)/dt effects not through NBCe1-A, but endogenous NHEs. Theoretical considerations argue against a CA stimulation ofHCO3-transport, supporting the conclusion that an NBCe1-A-HCO3-metabolon does not exist in oocytes.