Carbonic anhydrases enhance activity of endogenous Na-H exchangers and not the electrogenic Na/HCO cotransporter NBCe1-A, expressed in Xenopus oocytes.

Key Points: According to the metabolon hypothesis, direct association of cytosolic carbonic anhydrases (CAs) with the electrogenic Na/HCO cotransporter NBCe1-A speeds transport by regenerating/consuming . The present work addresses published discrepancies as to whether cytosolic CAs stimulate NBCe1-A, heterologously expressed in Xenopus oocytes. We confirm the essential elements of the previous experimental observations, taken as support for the 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- transporters (e.g. Na-H exchangers) by accelerating CO / -mediated buffering of acid-base equivalents, they could not appreciably affect transport rates of NBCe1 or other transporters carrying , , or ion pairs.

Abstract: The metabolon hypothesis predicts that cytosolic carbonic anhydrase (CA) binds to NBCe1-A, promotes replenishment/consumption, and enhances transport. Using a short step-duration current-voltage (I-V) protocol with Xenopus oocytes 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 ), which is a direct measure of NBCe1-A activity. Others - using bovine CA (bCA), untagged NBCe1-A, and protocols keeping holding potential (V ) far from NBCe1-A's reversal potential (E ) for prolonged periods - found that bCA increases total membrane current (ΔI ), 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, CO / exposures markedly decrease E , producing large transient outward currents persisting for >10 min and rapid increases in [Na ] . Although the CA inhibitor ethoxzolamide (EZA) reduces both ΔI and d[Na ] /dt, it does not reduce G . In oocytes not expressing NBCe1-A, CO / triggers rapid increases in [Na ] that both hCA II and bCA enhance in concentration-dependent manners. These d[Na ] /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 ΔI and d[Na ] /dt. Thus, CAs/EZA produce their ΔI and d[Na ] /dt effects not through NBCe1-A, but endogenous NHEs. Theoretical considerations argue against a CA stimulation of transport, supporting the conclusion that an NBCe1-A- metabolon does not exist in oocytes.