Facilitation by intracellular carbonic anhydrase of Na+-HCO3 - co-transport but not Na+/H+ exchange activity in the mammalian ventricular myocyte
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...
| Main Authors: | , , , , , , , , |
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| Format: | Journal article |
| Language: | English |
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2014
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| _version_ | 1797089329676288000 |
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| author | Villafuerte, F Swietach, P Youm, J Ford, K Cardenas, R Supuran, C Cobden, P Rohling, M Vaughan-Jones, R |
| author_facet | Villafuerte, F Swietach, P Youm, J Ford, K Cardenas, R Supuran, C Cobden, P Rohling, M Vaughan-Jones, R |
| author_sort | Villafuerte, F |
| collection | OXFORD |
| description | 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. © 2013 The Authors. The Journal of Physiology published by John Wiley and Sons Ltd on behalf of The Physiological Society. |
| first_indexed | 2024-03-07T03:02:33Z |
| format | Journal article |
| id | oxford-uuid:b17096a2-5595-4c68-b4d2-ac07fda1a4ed |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T03:02:33Z |
| publishDate | 2014 |
| record_format | dspace |
| spelling | oxford-uuid:b17096a2-5595-4c68-b4d2-ac07fda1a4ed2022-03-27T04:04:09ZFacilitation by intracellular carbonic anhydrase of Na+-HCO3 - co-transport but not Na+/H+ exchange activity in the mammalian ventricular myocyteJournal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:b17096a2-5595-4c68-b4d2-ac07fda1a4edEnglishSymplectic Elements at Oxford2014Villafuerte, FSwietach, PYoum, JFord, KCardenas, RSupuran, CCobden, PRohling, MVaughan-Jones, RCarbonic 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. © 2013 The Authors. The Journal of Physiology published by John Wiley and Sons Ltd on behalf of The Physiological Society. |
| spellingShingle | Villafuerte, F Swietach, P Youm, J Ford, K Cardenas, R Supuran, C Cobden, P Rohling, M Vaughan-Jones, R Facilitation by intracellular carbonic anhydrase of Na+-HCO3 - co-transport but not Na+/H+ exchange activity in the mammalian ventricular myocyte |
| title | Facilitation by intracellular carbonic anhydrase of Na+-HCO3
- co-transport but not Na+/H+ exchange activity in the mammalian ventricular myocyte |
| title_full | Facilitation by intracellular carbonic anhydrase of Na+-HCO3
- co-transport but not Na+/H+ exchange activity in the mammalian ventricular myocyte |
| title_fullStr | Facilitation by intracellular carbonic anhydrase of Na+-HCO3
- co-transport but not Na+/H+ exchange activity in the mammalian ventricular myocyte |
| title_full_unstemmed | Facilitation by intracellular carbonic anhydrase of Na+-HCO3
- co-transport but not Na+/H+ exchange activity in the mammalian ventricular myocyte |
| title_short | Facilitation by intracellular carbonic anhydrase of Na+-HCO3
- co-transport but not Na+/H+ exchange activity in the mammalian ventricular myocyte |
| title_sort | facilitation by intracellular carbonic anhydrase of na hco3 co transport but not na h exchange activity in the mammalian ventricular myocyte |
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