Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC48
A non-enzymatic transport metabolon enhances lactate flux in astrocytes
H. M. Becker1, M. H. Stridh2, M. D. Alt2, S. Wittmann1, R. McKenna3, G. Wennemuth4, J. W. Deitmer2
1. Department of Zoology / Membrane Transport, University of Kaiserslautern, Kaiserslautern, Germany. 2. Department of General Zoology, University of Kaiserslautern, Kaiserslauten, Germany. 3. Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida, United States. 4. Department of Anatomy and Cell Biology, Saarland University, Homburg/Saar, Germany.
High-energy metabolites, such as lactate, pyruvate, and ketone bodies, are transported into and out of cells via monocarboxylate transporters (MCT, SLC16), of which 14 isoforms have been described. In the brain, astrocytes express MCT1 and 4, which have been suggested to be responsible for the export of lactate to provide neurons with the energy substrate during increased activity. We have recently shown, that carbonic anhydrase isoform 2 (CAII) can increase lactate-induced acid/base flux via MCT1 and 4, heterologously expressed in Xenopus oocytes, in a non-catalytic manner (1, 2, 3). In the present study, we tested whether carbonic anhydrase can facilitate lactate transport in astrocytes. Our experiments revealed, that knock-out of CAII induced a significant reduction in the rate of lactate-induced acidification in mouse cerebellar astrocytes, as measured by in situ live-cell imaging with the pH-sensitive fluorescent dye 2’,7’-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) in acute cerebellar slices of CAII+/+ and CAII-/- mice (B6.D2-Car2n/J). In contrast to the knock-out, blocking CA catalytic activity with 6-Ethoxy-2-benzothiazolsulfonamid (EZA, 10 µM) had no effect on H+ flux, suggesting a non-catalytic facilitation of transport activity by CAII. The data could be confirmed by uptake experiments in cultured astrocytes of NMRI mice: Knock-down of CAII and CAIV by siRNA led to a significant decrease in lactate flux, while inhibition of CA catalytic activity with EZA (30 µM) had no effect. To check for a direct interaction between MCT1 and CAII in astrocytes, we applied an in situ proximity ligation assay, which indicated close proximity (< 40 nm) of MCT1 and CAII as intrinsically expressed in cultured mouse astrocytes. Co-immunoprecipitation with CAII and the C-terminal of MCT1 (MCT1-CT), fused to GST, revealed direct binding between MCT1-CT and CAII. To identify the binding site for CAII, we carried out single site mutations of MCT1, heterologously expressed in Xenopus laevis oocytes. The interaction between MCT1 and CAII seems to be facilitated by a cluster of three glutamate residues in the C-terminal of MCT1. While catalytic activity of CAII is apparently not necessary for enhancement of MCT1 transport activity, removal of the intramolecular H+-shuttle, His64, in CAII abolished the interaction between the two proteins in Xenopus oocytes. Our results suggest that CAII, directly bound to MCT1, can facilitate lactate flux in astrocytes by acting as a “proton collecting antenna” for the transporter. By this mechanism CAII would facilitate proton movement at the pore of the transporter, suppressing the build-up of proton microdomains, and thereby increasing trans-membrane lactate flux in astrocytes. <br /> This work was funded by the Deutsche Forschungsgemeinschaft DFG (De 231/24-1) and the “Research Initiative Membrane Biology”.
Where applicable, experiments conform with Society ethical requirements