Carbonic anhydrases enhance activity of endogenous Na-H exchangers and not the electrogenic Na/HCO3 cotransporter NBCe1-A, expressed in Xenopus oocytes

Abstract

Key points: According to the ${\mathrm{HCO}}_3^{-}$ metabolon hypothesis, direct association of cytosolic carbonic anhydrases (CAs) with the electrogenic Na/HCO₃ cotransporter NBCe1-A speeds transport by regenerating/consuming ${\mathrm{HCO}}_3^{-}$ . 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 ${\mathrm{HCO}}_3^{-}$ 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- ${\mathrm{HCO}}_3^{-}$ transporters (e.g. Na-H exchangers) by accelerating CO₂ / ${\mathrm{HCO}}_3^{-}$ -mediated buffering of acid-base equivalents, they could not appreciably affect transport rates of NBCe1 or other transporters carrying ${\mathrm{HCO}}_3^{-}$ , ${\mathrm{CO}}_3^{=}$ , or ${\mathrm{NaCO}}_3^{-}$ ion pairs.

Abstract: The ${\mathrm{HCO}}_3^{-}$ metabolon hypothesis predicts that cytosolic carbonic anhydrase (CA) binds to NBCe1-A, promotes ${\mathrm{HCO}}_3^{-}$ 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_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 (ΔI_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 / ${\mathrm{HCO}}_3^{-}$ exposures markedly decrease E_rev , producing large transient outward currents persisting for >10 min and rapid increases in [Na⁺ ]_i . Although the CA inhibitor ethoxzolamide (EZA) reduces both ΔI_m and d[Na⁺ ]_i /dt, it does not reduce G_NBC . In oocytes not expressing NBCe1-A, CO₂ / ${\mathrm{HCO}}_3^{-}$ 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 ΔI_m and d[Na⁺ ]_i /dt. Thus, CAs/EZA produce their ΔI_m and d[Na⁺ ]_i /dt effects not through NBCe1-A, but endogenous NHEs. Theoretical considerations argue against a CA stimulation of ${\mathrm{HCO}}_3^{-}$ transport, supporting the conclusion that an NBCe1-A- ${\mathrm{HCO}}_3^{-}$ metabolon does not exist in oocytes.

Keywords: NBCe1-A; NHE; SLC4A4; bicarbonate; carbonic anhydrase; metabolon.