title: Facilitation by intracellular carbonic anhydrase of Na+ -HCO3- co-transport but not Na+ / H+ exchange activity in the mammalian ventricular myocyte.
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.
authors.affiliation: Burdon Sanderson Cardiac Science Centre, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT, UK.  richard.vaughan-jones@dpag.ox.ac.uk.
authors.forename: Francisco C
authors.initials: FC
authors.lastname: Villafuerte
authors.forename: Pawel
authors.initials: P
authors.lastname: Swietach
authors.forename: Jae-Boum
authors.initials: JB
authors.lastname: Youm
authors.forename: Kerrie
authors.initials: K
authors.lastname: Ford
authors.forename: Rosa
authors.initials: R
authors.lastname: Cardenas
authors.forename: Claudiu T
authors.initials: CT
authors.lastname: Supuran
authors.forename: Philip M
authors.initials: PM
authors.lastname: Cobden
authors.forename: Mala
authors.initials: M
authors.lastname: Rohling
authors.forename: Richard D
authors.initials: RD
authors.lastname: Vaughan-Jones
mesh_terms: D000818:Animals; D001639:Bicarbonates / Q000737:chemistry / Q000378:metabolism*; D002256:Carbonic Anhydrases / Q000378:metabolism*; D002478:Cells, Cultured; D004789:Enzyme Activation; D006352:Heart Ventricles / Q000166:cytology / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008297:Male; D032383:Myocytes, Cardiac / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012964:Sodium / Q000378:metabolism*; D027982:Sodium-Bicarbonate Symporters / Q000378:metabolism*; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*
chemical_list: D001639:Bicarbonates; D027982:Sodium-Bicarbonate Symporters; D017923:Sodium-Hydrogen Exchangers; D012964:Sodium; D002256:Carbonic Anhydrases