Universität zu Köln

Al-Samir, Samer, Papadopoulos, Symeon, Scheibe, Renate J., Meissner, Joachim D., Cartron, Jean-Pierre, Sly, William S., Alper, Seth L., Gros, Gerolf and Endeward, Volker (2013). Activity and distribution of intracellular carbonic anhydrase II and their effects on the transport activity of anion exchanger AE1/SLC4A1. J. Physiol.-London, 591 (20). S. 4963 - 4983. HOBOKEN: WILEY. ISSN 1469-7793

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Abstract

We have investigated the previously published 'metabolon hypothesis' postulating that a close association of the anion exchanger 1 (AE1) and cytosolic carbonic anhydrase II (CAII) exists that greatly increases the transport activity of AE1. We study whether there is a physical association of and direct functional interaction between CAII and AE1 in the native human red cell and in tsA201 cells coexpressing heterologous fluorescent fusion proteins CAII-CyPet and YPet-AE1. In these doubly transfected tsA201 cells, YPet-AE1 is clearly associated with the cell membrane, whereas CAII-CyPet is homogeneously distributed throughout the cell in a cytoplasmic pattern. Forster resonance energy transfer measurements fail to detect close proximity of YPet-AE1 and CAII-CyPet. The absence of an association of AE1 and CAII is supported by immunoprecipitation experiments using Flag-antibody against Flag-tagged AE1 expressed in tsA201 cells, which does not co-precipitate native CAII but co-precipitates coexpressed ankyrin. Both the CAII and the AE1 fusion proteins are fully functional in tsA201 cells as judged by CA activity and by cellularHCO(3)(-) permeability (P-HCO3-) sensitive to inhibition by 4,4'-Diisothiocyano-2,2'-stilbenedisulfonic acid. Expression of the non-catalytic CAII mutant V143Y leads to a drastic reduction of endogenous CAII and to a corresponding reduction of total intracellular CA activity. Overexpression of an N-terminally truncated CAII lacking the proposed site of interaction with the C-terminal cytoplasmic tail of AE1 substantially increases intracellular CA activity, as does overexpression of wild-type CAII. These variously co-transfected tsA201 cells exhibit a positive correlation between cellular P-HCO3- and intracellular CA activity. The relationship reflects that expected from changes in cytoplasmic CA activity improving substrate supply to or removal from AE1, without requirement for a CAII-AE1 metabolon involving physical interaction. A functional contribution of the hypothesized CAII-AE1 metabolon to erythroid AE1-mediated HCO3- transport was further tested in normal red cells and red cells from CAII-deficient patients that retain substantial CA activity associated with the erythroid CAI protein lacking the proposed AE1-binding sequence. Erythroid P-HCO3- was indistinguishable in these two cell types, providing no support for the proposed functional importance of the physical interaction of CAII and AE1. A theoretical model predicts that homogeneous cytoplasmic distribution of CAII is more favourable for cellular transport of HCO3- and CO2 than is association of CAII with the cytoplasmic surface of the plasma membrane. This is due to the fact that the relatively slow intracellular transport of H+ makes it most efficient to place the CA in the vicinity of the haemoglobin molecules, which are homogeneously distributed over the cytoplasm.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Al-Samir, Samer UNSPECIFIED UNSPECIFIED UNSPECIFIED Papadopoulos, Symeon UNSPECIFIED UNSPECIFIED UNSPECIFIED Scheibe, Renate J. UNSPECIFIED UNSPECIFIED UNSPECIFIED Meissner, Joachim D. UNSPECIFIED UNSPECIFIED UNSPECIFIED Cartron, Jean-Pierre UNSPECIFIED UNSPECIFIED UNSPECIFIED Sly, William S. UNSPECIFIED UNSPECIFIED UNSPECIFIED Alper, Seth L. UNSPECIFIED UNSPECIFIED UNSPECIFIED Gros, Gerolf UNSPECIFIED UNSPECIFIED UNSPECIFIED Endeward, Volker UNSPECIFIED UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-473986
DOI:	10.1113/jphysiol.2013.251181
Journal or Publication Title:	J. Physiol.-London
Volume:	591
Number:	20
Page Range:	S. 4963 - 4983
Date:	2013
Publisher:	WILEY
Place of Publication:	HOBOKEN
ISSN:	1469-7793
Language:	English
Faculty:	Unspecified
Divisions:	Unspecified
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language CO2 PERMEABILITY; PROTON MOBILITY; BINDING-SITE; MEMBRANE; PROTEIN; COTRANSPORTER; DIFFUSION; METABOLON; MECHANISM; BAND-3 Multiple languages Neurosciences; Physiology Multiple languages
URI:	http://kups.ub.uni-koeln.de/id/eprint/47398