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Facilitation by intracellular carbonic anhydrase of Na(+)–HCO(3)(−) co-transport but not Na(+)/H(+) exchange activity in the mammalian ventricular myocyte

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 lo...

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Autores principales: Villafuerte, Francisco C, Swietach, Pawel, Youm, Jae-Boum, Ford, Kerrie, Cardenas, Rosa, Supuran, Claudiu T, Cobden, Philip M, Rohling, Mala, Vaughan-Jones, Richard D
Formato: Online Artículo Texto
Lenguaje:English
Publicado: John Wiley & Sons Ltd 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3948559/
https://www.ncbi.nlm.nih.gov/pubmed/24297849
http://dx.doi.org/10.1113/jphysiol.2013.265439
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author Villafuerte, Francisco C
Swietach, Pawel
Youm, Jae-Boum
Ford, Kerrie
Cardenas, Rosa
Supuran, Claudiu T
Cobden, Philip M
Rohling, Mala
Vaughan-Jones, Richard D
author_facet Villafuerte, Francisco C
Swietach, Pawel
Youm, Jae-Boum
Ford, Kerrie
Cardenas, Rosa
Supuran, Claudiu T
Cobden, Philip M
Rohling, Mala
Vaughan-Jones, Richard D
author_sort Villafuerte, Francisco C
collection PubMed
description 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 HCO(3)(−) transport, and hence pH(i) regulation in the heart.
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spelling pubmed-39485592014-05-22 Facilitation by intracellular carbonic anhydrase of Na(+)–HCO(3)(−) co-transport but not Na(+)/H(+) exchange activity in the mammalian ventricular myocyte Villafuerte, Francisco C Swietach, Pawel Youm, Jae-Boum Ford, Kerrie Cardenas, Rosa Supuran, Claudiu T Cobden, Philip M Rohling, Mala Vaughan-Jones, Richard D J Physiol Cardiovascular 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 HCO(3)(−) transport, and hence pH(i) regulation in the heart. John Wiley & Sons Ltd 2014-03-01 2014-01-09 /pmc/articles/PMC3948559/ /pubmed/24297849 http://dx.doi.org/10.1113/jphysiol.2013.265439 Text en © 2013 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society http://creativecommons.org/licenses/by/3.0/ This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
spellingShingle Cardiovascular
Villafuerte, Francisco C
Swietach, Pawel
Youm, Jae-Boum
Ford, Kerrie
Cardenas, Rosa
Supuran, Claudiu T
Cobden, Philip M
Rohling, Mala
Vaughan-Jones, Richard D
Facilitation by intracellular carbonic anhydrase of Na(+)–HCO(3)(−) co-transport but not Na(+)/H(+) exchange activity in the mammalian ventricular myocyte
title Facilitation by intracellular carbonic anhydrase of Na(+)–HCO(3)(−) co-transport but not Na(+)/H(+) exchange activity in the mammalian ventricular myocyte
title_full Facilitation by intracellular carbonic anhydrase of Na(+)–HCO(3)(−) co-transport but not Na(+)/H(+) exchange activity in the mammalian ventricular myocyte
title_fullStr Facilitation by intracellular carbonic anhydrase of Na(+)–HCO(3)(−) co-transport but not Na(+)/H(+) exchange activity in the mammalian ventricular myocyte
title_full_unstemmed Facilitation by intracellular carbonic anhydrase of Na(+)–HCO(3)(−) co-transport but not Na(+)/H(+) exchange activity in the mammalian ventricular myocyte
title_short Facilitation by intracellular carbonic anhydrase of Na(+)–HCO(3)(−) co-transport but not Na(+)/H(+) exchange activity in the mammalian ventricular myocyte
title_sort facilitation by intracellular carbonic anhydrase of na(+)–hco(3)(−) co-transport but not na(+)/h(+) exchange activity in the mammalian ventricular myocyte
topic Cardiovascular
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3948559/
https://www.ncbi.nlm.nih.gov/pubmed/24297849
http://dx.doi.org/10.1113/jphysiol.2013.265439
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