Facilitation by intracellular carbonic anhydrase of Na+–HCO3− co‐transport but not Na+/H+ exchange activity in the mammalian ventricular myocyte
Authors: Villafuerte Francisco C. , Swietach Pawel , Youm Jae‐Boum , Ford Kerrie , Cardenas Rosa , Supuran Claudiu T. , Cobden Philip M. , Rohling Mala , Vaughan‐Jones Richard D. 
Published: December 2013

Key points
Carbonic anhydrase enzymes are said to enhance membrane H+ and HCO3− transport, an idea tested almost exclusively in heterologous cell‐expression systems.
In ventricular myocytes, inhibiting cytoplasmic enzyme activity slows membrane acid extrusion (HCO3− influx) on Na+–HCO3− co‐transporters, with no effect on Na+/H+ exchangers. Inhibiting exofacial enzyme activity has no effect on either transporter.
Mathematical modelling simulates the influence of carbonic anhydrase on Na+–HCO3− co‐transport, provided the enzyme catalytically delivers H+ ions to the transporter (across a cytoplasmic nanodomain that is poorly accessible to intrinsic buffers), rather than catalysing protonation of the imported bicarbonate. Intrinsic cytoplasmic mobile buffers appear to deliver H+ to Na+/H+ exchangers, thus obviating the need for carbonic anhydrase.
Conclusion: in native cardiac cells, intracellular carbonic anhydrase molecules partner Na+–HCO3− co‐transporters but not Na+/H+ exchangers, to enhance flux activity. The enzyme thus plays a key role in supporting bicarbonate transport and pH control in the heart.
AbstractCarbonic 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 (CAi and CAe) 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 CAi activity alone. Results are quantitatively consistent with a model where CAi 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 CAi 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.