Functional consequences of engineering the hydrophobic pocket of carbonic anhydrase II

؊ exchange by an AE1 missense mutant devoid of CA2 binding, with activity further enhanced by CA2 co-expression. The same transport-incompetent AE1 mutants failed to rescue Cl ؊ /HCO 3 ؊ exchange by the AE1 truncation mutant 896X, despite preservation of the latter's core CA2 binding site. These data increase the minimal extent of a functionally defined CA2 binding site in AE1. The inter-protomeric rescue of HCO 3 ؊ transport within the AE1 dimer shows functional proximity of the C-terminal cytoplasmic tail of one protomer to the anion translocation pathway in the adjacent protomer within the AE1 heterodimer. The data strongly support the hypothesis that an intact transbilayer anion translocation pathway is completely contained within an AE1 monomer.

Section: Functional Effects Of Sequential Truncation Of the Kae1 C-mentioning

confidence: 82%

Deficient HCO3- Transport in an AE1 Mutant with Normal Cl- Transport Can be Rescued by Carbonic Anhydrase II Presented on an Adjacent AE1 Protomer

Dahl¹,

Jiang

Chernova

et al. 2003

“…V143Y CAII has 3000-fold lower catalytic activity than wild-type CAII (34). Although this is a great reduction, the catalytic rate of ϳ300 s Ϫ1 remains.…”

Section: Resultsmentioning

confidence: 98%

“…The XbaI-EcoRI fragment was then cloned into XbaI/EcoRI-digested pRBG4 vector to yield pJRC36 (37). An expression construct for the V143Y CAII mutant called pDS14 was constructed by the same strategy using V143Y cDNA supplied by Dr. Carol Fierke (34). The construct was verified by DNA sequencing performed by the Core Facility in the Department of Biochemistry, University of Alberta with an Applied Biosystems 373A DNA sequencer.…”

Section: Methodsmentioning

confidence: 99%

A Transport Metabolon

Sterling

Reithmeier

Casey

2001

336

153

The cytoplasmic carboxyl-terminal domain of AE1, the plasma membrane chloride/bicarbonate exchanger of erythrocytes, contains a binding site for carbonic anhydrase II (CAII). To examine the physiological role of the AE1/CAII interaction, anion exchange activity of transfected HEK293 cells was monitored by following the changes in intracellular pH associated with AE1-mediated bicarbonate transport. AE1-mediated chloride/bicarbonate exchange was reduced 50 -60% by inhibition of endogenous carbonic anhydrase with acetazolamide, which indicates that CAII activity is required for full anion transport activity. AE1 mutants, unable to bind CAII, had significantly lower transport activity than wild-type AE1 (10% of wild-type activity), suggesting that a direct interaction was required. To determine the effect of displacement of endogenous wild-type CAII from its binding site on AE1, AE1-transfected HEK293 cells were co-transfected with cDNA for a functionally inactive CAII mutant, V143Y. AE1 activity was maximally inhibited 61 ؎ 4% in the presence of V143Y CAII. A similar effect of V143Y CAII was found for AE2 and AE3cardiac anion exchanger isoforms. We conclude that the binding of CAII to the AE1 carboxyl-terminus potentiates anion transport activity and allows for maximal transport. The interaction of CAII with AE1 forms a transport metabolon, a membrane protein complex involved in regulation of bicarbonate metabolism and transport.Carbon dioxide, the metabolic end product of oxidative respiration, must be effectively cleared from the human body. CO 2 diffuses out of cells into the blood stream and into erythrocytes, where it is hydrated by cytosolic carbonic anhydrase (CA). 1 The resulting membrane-impermeant HCO 3 Ϫ is exported into the plasma by the plasma membrane Cl Ϫ /HCO 3 Ϫ anion exchanger (AE1), thus increasing the blood capacity for carrying CO 2 . Upon returning to the lungs the process is reversed; HCO 3 Ϫ is transported into the erythrocyte in exchange for Cl Ϫ by AE1 and dehydrated by CA, and the resulting CO 2 diffuses across the erythrocyte and alveolar membranes to be expired from the body. The 5 ϫ 10 4 s Ϫ1 turnover rate of AE1 (1) and the high content of AE1 in the membrane (2) facilitate completion of bicarbonate transport within 50 ms during passage of an erythrocyte through a capillary (3).AE1 is a 911-amino acid polytopic glycoprotein that facilitates the one for one electroneutral exchange of Cl Ϫ for HCO 3

“…The catalytically inactive mutant CAII-V143Y was a gift from Dr. Carol Fierke, Ann Arbor, MI (26,27). The two constructs were subcloned into the oocyte expression vector pGEM-He-Juel, which contains the 5Ј and the 3Ј untranscribed regions of the Xenopus ␤-globulin flanking the multiple cloning site.…”

Section: Methodsmentioning

confidence: 99%

Nonenzymatic Proton Handling by Carbonic Anhydrase II during H+-Lactate Cotransport via Monocarboxylate Transporter 1

Becker

Deitmer

2008

105

Carbonic anhydrase (CA) is a ubiquitous enzyme catalyzing the equilibration of carbon dioxide, protons, and bicarbonate. For several acid/base-coupled membrane carriers it has been shown that the catalytic activity of CA supports transport activity, an interaction coined "transport metabolon." We have reported that CA isoform II (CAII) enhances lactate transport activity of the monocarboxylate transporter isoform I (MCT1) expressed in Xenopus oocytes, which does not require CAII catalytic activity (Becker, H. M., Fecher-Trost, C., Hirnet, D., Sült-emeyer, D., and Deitmer, J. W. (2005) J. Biol. Chem. 280, 39882-39889). Coexpression of MCT1 with either wild type CAII or the catalytically inactive mutant CAII-V143Y similarly enhanced MCT1 activity, although injection of CAI or coexpression of an N-terminal mutant of CAII had no effect on MCT1 transport activity, demonstrating a specific, nonenzymatic action of CAII on lactate transport via MCT1. If the H ؉ gradient was set to dominate the rate of lactate transport by applying low concentrations of lactate at a high H ؉ concentration, the effect of CAII was largest. We tested the hypothesis of whether CAII helps to shuttle H ؉ along the inner face of the cell membrane by measuring the pH change with fluorescent dye in different areas of interest during focal lactate application. Intracellular pH shifts decayed from the focus of lactate application to more distant sites much less when CAII had been injected. We present a hypothetical model in which the effective movement of H ؉ into the bulk cytosol is increased by CAII, thus slowing the dissipation of the H ؉ gradient across the cell membrane, which drives MCT1 activity.Transport of acid/base equivalents across cell membranes plays a crucial role both for pH regulation and cellular import and export of metabolites. Many Na ϩ -dependent and Na ϩ -independent transport modes of metabolites (e.g. neurotransmitter substances) also transport H ϩ , OH Ϫ , or HCO 3 Ϫ as co-or counter-substrate. The energetic compounds lactate and pyruvate are primarily transported by monocarboxylate transporters (MCT) 2 in an electroneutral 1 proton-1 organic anion transport mode. Members of the MCT family, which belong to the human gene family SLC16 which comprise 14 isoforms, are expressed in most tissues, in particular those with a large energy consumption like muscle and brain (1, 2). In the brain MCT1 is located mainly in glial cells, where it facilitates the export of lactate, which is then taken up by neurons via the high affinity MCT2; thereby the two isoforms are believed to shuttle lactate from glial cells to neurons which seems to be pivotal for brain energy metabolism (3-6). In muscle, MCT1 is highly expressed in oxidative type I fibers, where it mediates import of lactate that is released by glycolytic muscle cells via MCT3 and MCT4 (7-9). MCT1, when expressed in Xenopus oocytes, operates with a K m value of 3.5 mM for lactate influx and of 6.4 mM for efflux of lactate. Both the rate and the amplitude of the lactate-induced ac...