Abstract
Key points: According to the HCO-3<math><msubsup><mi>HCO</mi><mn>3</mn><mo>-</mo></msubsup></math> metabolon hypothesis, direct association of cytosolic carbonic anhydrases (CAs) with the electrogenic Na/HCO3 cotransporter NBCe1-A speeds transport by regenerating/consuming HCO-3<math><msubsup><mi>HCO</mi><mn>3</mn><mo>-</mo></msubsup></math> . 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 HCO-3<math><msubsup><mi>HCO</mi><mn>3</mn><mo>-</mo></msubsup></math> 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- HCO-3<math><msubsup><mi>HCO</mi><mn>3</mn><mo>-</mo></msubsup></math> transporters (e.g. Na-H exchangers) by accelerating CO2 / HCO-3<math><msubsup><mi>HCO</mi><mn>3</mn><mo>-</mo></msubsup></math> -mediated buffering of acid-base equivalents, they could not appreciably affect transport rates of NBCe1 or other transporters carrying HCO-3<math><msubsup><mi>HCO</mi><mn>3</mn><mo>-</mo></msubsup></math> , CO=3<math><msubsup><mi>CO</mi><mn>3</mn><mo>=</mo></msubsup></math> , or NaCO-3<math><msubsup><mi>NaCO</mi><mn>3</mn><mo>-</mo></msubsup></math> ion pairs. The HCO-3<math><msubsup><mi>HCO</mi><mn>3</mn><mo>-</mo></msubsup></math> metabolon hypothesis predicts that cytosolic carbonic anhydrase (CA) binds to NBCe1-A, promotes HCO-3<math><msubsup><mi>HCO</mi><mn>3</mn><mo>-</mo></msubsup></math> 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 (GNBC ), which is a direct measure of NBCe1-A activity. Others - using bovine CA (bCA), untagged NBCe1-A, and protocols keeping holding potential (Vh ) far from NBCe1-A's reversal potential (Erev ) for prolonged periods - found that bCA increases total membrane current (ΔIm ), 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 / HCO-3<math><msubsup><mi>HCO</mi><mn>3</mn><mo>-</mo></msubsup></math> exposures markedly decrease Erev , producing large transient outward currents persisting for >10 min and rapid increases in [Na+ ]i . Although the CA inhibitor ethoxzolamide (EZA) reduces both ΔIm and d[Na+ ]i /dt, it does not reduce GNBC . In oocytes not expressing NBCe1-A, CO2 / HCO-3<math><msubsup><mi>HCO</mi><mn>3</mn><mo>-</mo></msubsup></math> 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 ΔIm and d[Na+ ]i /dt. Thus, CAs/EZA produce their ΔIm and d[Na+ ]i /dt effects not through NBCe1-A, but endogenous NHEs. Theoretical considerations argue against a CA stimulation of HCO-3<math><msubsup><mi>HCO</mi><mn>3</mn><mo>-</mo></msubsup></math> transport, supporting the conclusion that an NBCe1-A- HCO-3<math><msubsup><mi>HCO</mi><mn>3</mn><mo>-</mo></msubsup></math> metabolon does not exist in oocytes.