Voltage Dependence of H+ Buffering Mediated by Sodium Bicarbonate Cotransport Expressed in Xenopus Oocytes

Becker, Holger M.; Deitmer, Joachim W.

doi:10.1074/jbc.m402401200

Cited by 38 publications

(29 citation statements)

References 21 publications

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“…Indeed, Kristensen et al (26) have pointed out that this isoform was clearly present in different rat skeletal muscles, but without any correlation to fiber type. In addition, an NBC construct derived from the kidney corresponding to this isoform was also used by Becker and colleagues (3,4) in their in vitro studies to demonstrate the functional cooperation between NBC and MCT1. The NBC content was quantified by isoform-nonspecific NBC antibodies as previously used by Juel et al (25) and Kristensen et al (26) in skeletal muscle (NBC antibody, AF04017; Chemicon International 3212).…”

Section: Transport Protein Expressionmentioning

confidence: 99%

Effects of high-intensity training on MCT1, MCT4, and NBC expressions in rat skeletal muscles: influence of chronic metabolic alkalosis

Thomas¹,

Bishop²,

Moore-Morris³

et al. 2007

American Journal of Physiology-Endocrinology and Metabolism

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Section: Transport Protein Expressionmentioning

confidence: 99%

Effects of high-intensity training on MCT1, MCT4, and NBC expressions in rat skeletal muscles: influence of chronic metabolic alkalosis

Thomas¹,

Bishop²,

Moore-Morris³

et al. 2007

American Journal of Physiology-Endocrinology and Metabolism

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“…To increase the opening of the electrode tip, it was bevelled with a jet stream of aluminum powder suspended in H 2 O. Calibration of the electrodes was carried out in OR2ϩ with a pH of 7.0 and 6.4. The recording arrangement was the same as described previously (19,20). The central and the reference barrel of the electrodes were connected by chlorided silver wires to the head stages of an electrometer amplifier.…”

Section: Constructs Oocytes and Injection Of Crna And Carbonicmentioning

confidence: 99%

“…This was achieved by carefully rotating the oocyte with the impaled electrode. All experiments were carried out at room temperature (22-25°C (20).…”

Section: Constructs Oocytes and Injection Of Crna And Carbonicmentioning

confidence: 99%

Transport Activity of MCT1 Expressed in Xenopus Oocytes Is Increased by Interaction with Carbonic Anhydrase

Becker

Hirnet²,

Fecher‐Trost³

et al. 2005

Journal of Biological Chemistry

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Injection of carbonic anhydrase isoform II (CA) into Xenopus frog oocytes increased the rate of H ؉ flux via the rat monocarboxylate transporter isoform 1 (MCT1) expressed in the oocytes. MCT1 activity was assessed by changes of intracellular H ؉ concentration measured by pH-selective microelectrodes during application of lactate. CA-induced augmentation of the rate of H ؉ flux mediated by MCT1 was not inhibited by ethoxyzolamide (10 M) and did not depend on the presence of added CO 2 /HCO 3 ؊ but was suppressed by injection of an antibody against CA. Deleting the C terminus of the MCT1 greatly reduced its transport rate and removed transport facilitation by CA. Injected CA accelerated the CO 2 /HCO 3 ؊ -induced acidification severalfold, which was blocked by ethoxyzolamide and was independent of MCT1 expression. Mass spectrometry confirmed activity of CA as injected into the frog oocytes. With pulldown assays we demonstrated a specific binding of CA to MCT1 that was not attributed to the C terminus of MCT1. Our results suggest that CA enhances MCT1 transport activity, independent of its enzymatic reaction center, presumably by binding to MCT1.Acid/base transport across cell membranes includes a variety of carriers, some of which are instrumental for cellular pH regulation or cellular import and export of metabolites. The monocarboxylate transporters (MCT) 2 belong to the SLC16 human gene family, and 14 isoforms have been described so far for this lactate/pyruvate-proton cotransporter (1). MCT isoforms have been reported for most tissues, where the removal or supply of L-lactate (pyruvate or other monocarboxylates) is related to different metabolic conditions. The MCT isoform 1 (MCT1) has been found in most tissues; in the brain, MCT1 appears to be located mainly in glial cells (2). MCT1 has been expressed in oocytes of the frog Xenopus laevis, where it has been characterized molecularly and functionally (2-4). MCT1 has a K m value for L-lactate of 3-5 mM and is believed to be associated in the glia-neuron lactate shuttle (5, 6). Lactate is exported from astrocytes via MCT1 and is then taken up by neurons via the high-affinity MCT2 (K m ϳ0.5 mM). The significance of this lactate shuttle has been demonstrated by showing that synaptic activity recovers and is maintained under glucose-deprived conditions after addition of lactate (7-9).In the present study we have looked for a possible role for carbonic anhydrase (CA) in H ϩ fluxes via MCT1. Extracellular CA isoform IV has been shown to facilitate lactate transport in astrocytes and neurons (10). Furthermore, CA has been reported to bind to various acid/base transporters, such as the chloride/bicarbonate anion exchanger (11), the sodium bicarbonate cotransporter (12, 13), and the sodium/hydrogen exchanger (14). We have expressed MCT1 in Xenopus oocytes and injected isoform II of CA, which has been shown to be present also in astrocytes (15). Our results suggest that CA binds to the MCT1 in the cell membrane of the oocyte and greatly enhances the H ϩ flux via the MC...

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“…Both the rate and the amplitude of the lactate-induced acidification strongly depend on the extracellular H ϩ concentration (10,11). Expressed in frog oocytes, MCT1 has been shown to cooperate with the sodium bicarbonate cotransporter NBCe1 (12,13) and with carbonic anhydrase isoform II (CAII) (14). Remarkably, the support of MCT1 activity by CAII was independent of the catalytic activity of the enzyme, which catalyzes the equilibrium of CO 2 , H ϩ , and HCO 3 Ϫ (14).…”

mentioning

confidence: 99%

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

Becker

Deitmer

2008

Journal of Biological Chemistry

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105

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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...

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Voltage Dependence of H+ Buffering Mediated by Sodium Bicarbonate Cotransport Expressed in Xenopus Oocytes

Cited by 38 publications

References 21 publications

Effects of high-intensity training on MCT1, MCT4, and NBC expressions in rat skeletal muscles: influence of chronic metabolic alkalosis

Effects of high-intensity training on MCT1, MCT4, and NBC expressions in rat skeletal muscles: influence of chronic metabolic alkalosis

Transport Activity of MCT1 Expressed in Xenopus Oocytes Is Increased by Interaction with Carbonic Anhydrase

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

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