Transport activity of MCT1 expressed in Xenopus oocytes is increased by interaction with carbonic anhydrase
ABSTRACT 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 microM) and did not depend on the presence of added CO2/HCO3- 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 CO2/HCO3(-)-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.
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ABSTRACT: Colorectal cancer (CRC) is one of the most common solid tumors worldwide. A diet rich in dietary fiber is associated with a reduction in its risk. Butyrate (BT) is one of the main end products of anaerobic bacterial fermentation of dietary fiber in the human colon. This short-chain fatty acid is an important metabolic substrate in normal colonic epithelial cells and has important homeostatic functions at this level, including the ability to prevent/inhibit carcinogenesis. BT is transported into colonic epithelial cells by two specific carrier-mediated transport systems, the monocarboxylate transporter 1 (MCT1) and the sodium-coupled monocarboxylate transporter 1 (SMCT1). In normal colonic epithelial cells, BT is the main energy source for normal colonocytes and it is effluxed by BCRP. Colonic epithelial tumoral cells show a reduction in BT uptake (through a reduction in MCT1 and SMCT1 protein expression), an increase in the rate of glucose uptake and glycolysis becomes their primary energy source. BT presents an anticarcinogenic effect (induction of cell differentiation and apoptosis and inhibition of cell proliferation) but has an apparent opposing effect upon growth of normal colonocytes (the "BT paradox"). Because the cellular effects of BT (e.g. inhibition of histone deacetylases) are dependent on its intracellular concentration, knowledge on the mechanisms involved in BT membrane transport and its regulation seem particularly relevant in the context of the physiological and pharmacological benefits of this compound. This review discusses the mechanisms of BT transport and integrates this knowledge with the effects of BT in tumoral and normal colonocytes.Current Drug Metabolism 11/2013; 14(9):994-1008. DOI:10.2174/1389200211314090006 · 3.49 Impact Factor
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ABSTRACT: Proton-coupled monocarboxylate transporters (MCTs) mediate the exchange of high-energy metabolites like lactate between different cells and tissues. We have reported previously that carbonic anhydrase II augments transport activity of MCT1 and MCT4 by a non-catalytic mechanism, while leaving transport activity of MCT2 unaltered. In the present study we combined electrophysiological measurements in Xenopus oocytes and pull-down experiments to analyze the direct interaction between CAII and MCT1, MCT2 and MCT4, respectively. Transport activity of MCT2-WT, which lacks a putative CAII-binding site, is not augmented by CAII. However, introduction of a CAII-binding site into the C-terminus of MCT2 resulted in CAII-mediated facilitation of MCT2 transport activity. Interestingly, introduction of three glutamic acid residues alone was not sufficient to establish a direct interaction between MCT2 and CAII, but the cluster had to be arranged in a fashion that allowed access to the binding moiety in CAII. We further demonstrate that functional interaction between MCT4 and CAII requires direct binging of the enzyme to the acidic cluster E431EE in the C-terminus of MCT4 in a similar fashion as previously shown for binding of CAII to the cluster E489EE in the C-terminus of MCT1. In CAII, binding to MCT1 and MCT4 is mediated by a histidine residue at position 64. Taken together, our results suggest that facilitation of MCT transport activity by CAII requires direct binding between histidine 64 in CAII and a cluster of glutamic acid residues in the transporter's C-terminus that has to be positioned in a surrounding that allows access to CAII. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.Journal of Biological Chemistry 01/2015; 290(7). DOI:10.1074/jbc.M114.624577 · 4.60 Impact Factor
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ABSTRACT: Acidosis in the brain may severely impair a variety of functions, including synap-tic transmission, metabolic energy supply, membrane transport and other processes (Ruusuvuori and Kaila, 2014). Transport of acid–base equivalents across the cell membrane of neurons and glial cells also results in pH changes in the extracellular spaces. Cytosolic and extracellular buffer capacity and the activ-ity of carbonic anhydrases contribute to shape pH changes, which can be elicited by neuronal activity, neurotransmitters and neuromodulators, metabolic pro-cesses, active cellular pH regulation, and secondary transporters carrying acid– base equivalents, and in turn these pH changes can affect neuronal functions (Deitmer and Rose, 1996; Chesler, 2003). The free H + concentration in cells is in the nanomolar range, and the high buffer capacity of cells provides a reservoir of acid equivalents in the millimolar range. In other words, there is a pool of protons in rapid exchange between buffer sites and free solution, with 10 5 or more protons being buffered for each proton in solution. At a blood pH of 7.4, and 7.2–7.3 in the extracellular spaces of brain tissue (Cragg et al., 1977; Ruusuvuori and Kaila, 2014), and with a negative membrane potential of between −50 and −90 mV in mammalian brain cells, H + has to be continuously extruded to maintain a physiological cytosolic pH of 7.0–7.3. Nevertheless, pH changes may peak well outside this range, at least for short time periods, and may be considered as H + signals, sometimes even with neurotransmitter function (Deitmer and Rose, 1996; Du et al., 2014). The net extrusion of acid from neurons and glial cells is accomplished by secondary active transport, wherein the efflux of H + or the influx of HCO − 3 is coupled to Na + influx, utilizing energy stored in the trans-membrane Na + gradient. pH regulation in these cells involves a variety of membrane acid–base carriers, including sodium– hydrogen exchange, sodium–bicarbonate cotransport, and sodium-dependent and sodium-independent chloride– bicarbonate exchange. In addition, there are a number of acid/base-coupled car-riers, which are linked to the transport of metabolites, such as lactate and amino acids. The lactate transport via mono-carboxylate transporters (MCTs) has been suggested to play a major role for the supply of energy to neurons, and led to the "Astrocyte-to-Neuron Lactate Shuttle Hypothesis" (ANLSH; Pellerin and Magistretti, 1994).