Facilitation by intracellular carbonic anhydrase of Na⁺-HCO₃^- co-transport but not Na⁺/H⁺ exchange activity in the mammalian ventricular myocyte

Carbonic anhydrase enzymes (CAs) catalyse the reversible hydration of CO₂ to H⁺ and HCO₃^- ions. This catalysis is proposed to be harnessed by acid/base transporters, to facilitate their transmembrane flux activity, either through direct protein-protein binding (a 'transport metabolon') or local functional interaction. Flux facilitation has previously been investigated by heterologous co-expression of relevant proteins in host cell lines/oocytes. Here, we examine the influence of intrinsic CA activity on membrane HCO₃^- or H⁺ transport via the native acid-extruding proteins, Na⁺-HCO₃^- cotransport (NBC) and Na⁺/H⁺ exchange (NHE), expressed in enzymically isolated mammalian ventricular myocytes. Effects of intracellular and extracellular (exofacial) CA (CA_i and CA_e) are distinguished using membrane-permeant and -impermeant pharmacological CA inhibitors, while measuring transporter activity in the intact cell using pH and Na⁺ fluorophores. We find that NBC, but not NHE flux is enhanced by catalytic CA activity, with facilitation being confined to CA_i activity alone. Results are quantitatively consistent with a model where CA_i catalyses local H⁺ ion delivery to the NBC protein, assisting the subsequent (uncatalysed) protonation and removal of imported HCO₃^- ions. In well-superfused myocytes, exofacial CA activity is superfluous, most likely because extracellular CO₂/HCO₃^- buffer is clamped at equilibrium. The CA_i insensitivity of NHE flux suggests that, in the native cell, intrinsic mobile buffer-shuttles supply sufficient intracellular H+ ions to this transporter, while intrinsic buffer access to NBC proteins is restricted. Our results demonstrate a selective CA facilitation of acid/base transporters in the ventricular myocyte, implying a specific role for the intracellular enzyme in HCO3 - transport, and hence pHi regulation in the heart.