Facilitation by intracellular carbonic anhydrase of Na(+)–HCO(3)(−) co-transport but not Na(+)/H(+) exchange activity in the mammalian ventricular myocyte

Carbonic anhydrase enzymes (CAs) catalyse the reversible hydration of CO(2) to H(+) and HCO(3)(−) 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 HCO(3)(−) or H(+) transport via the native acid-extruding proteins, Na(+)–HCO(3)(−) 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 HCO(3)(−) ions. In well-superfused myocytes, exofacial CA activity is superfluous, most likely because extracellular CO(2)/HCO(3)(−) 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 HCO(3)(−) transport, and hence pH(i) regulation in the heart.