Abstract

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 (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.

Highlights

  • Carbonic anhydrase (CA) proteins are expression products of a gene-family that codes for at least 16 isoforms (Sly & Hu, 1995; Supuran, 2008)

  • In the present work we have examined the influence of the catalytic activity of intracellular and exofacial CA (CAi and extracellular exofacial CA (CAe), respectively) on Na+/H+ exchange (NHE)- and Na+–HCO3− cotransport (NBC)-mediated acid efflux from rat ventricular myocytes

  • These results differ from previous work in heterologous expression systems where the flux activity of NHE1, NBCe1 and NBCn1, expressed in oocytes or various cell lines, has been suggested to be enhanced by the co-expression of either cytoplasmic CAII or exofacial CAIV (Li et al 2002, 2006; Alvarez et al 2003; Loiselle et al 2004; Becker & Deitmer, 2007; Schueler et al 2011), CA proteins that are endogenously expressed in the myocardium (Scheibe et al 2006)

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Summary

Introduction

Carbonic anhydrase (CA) proteins are expression products of a gene-family that codes for at least 16 isoforms (Sly & Hu, 1995; Supuran, 2008). A major role for CAs is proposed to be the catalytic enhancement of acid/base membrane transport. Some reports suggest they achieve this by binding directly to pH regulatory transporters, forming a multimeric protein complex called a transport metabolon (Sterling et al 2001, 2002; Morgan et al 2007; Casey et al 2009; Svastova et al 2012; Vargas & Alvarez, 2012). For example, CA protein may stimulate acid transport allosterically, even in the absence of enzymic activity (Becker et al 2011)

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