Facilitation by intracellular carbonic anhydrase of Na<sup>+</sup>-HCO<inf>3</inf><sup>-</sup> co-transport but not Na<sup>+</sup>/H<sup>+</sup> exchange activity in the mammalian ventricular myocyte
Villafuerte FC., Swietach P., Youm JB., Ford K., Cardenas R., Supuran CT., Cobden PM., Rohling M., Vaughan-Jones RD.
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 HCO3 - transport, and hence pHi regulation in the heart. © 2013 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.