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

J Physiol. 2014 Mar 1;592(5):991-1007. doi: 10.1113/jphysiol.2013.265439. Epub 2013 Dec 2.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Bicarbonates / chemistry
  • Bicarbonates / metabolism*
  • Carbonic Anhydrases / metabolism*
  • Cells, Cultured
  • Enzyme Activation
  • Heart Ventricles / cytology
  • Heart Ventricles / metabolism*
  • Hydrogen-Ion Concentration
  • Male
  • Myocytes, Cardiac / metabolism*
  • Rats
  • Rats, Sprague-Dawley
  • Sodium / metabolism*
  • Sodium-Bicarbonate Symporters / metabolism*
  • Sodium-Hydrogen Exchangers / metabolism*

Substances

  • Bicarbonates
  • Sodium-Bicarbonate Symporters
  • Sodium-Hydrogen Exchangers
  • Sodium
  • Carbonic Anhydrases