Proceedings of The Physiological Society

University of Oxford (2011) Proc Physiol Soc 23, PC64

Poster Communications

High-affinity lactate uptake is facilitated by an extracellular, but not by an intracellular carbonic anhydrase

M. Klier1,2, C. Schüler1, A. P. Halestrap3, W. S. Sly4, H. M. Becker2, J. W. Deitmer1

1. General Zoology, TU Kaiserslautern, Kaiserslautern, Germany. 2. Zoology / Membrane Transport, TU Kaiserslautern, Kaiserslautern, Germany. 3. School of Biochemistry and the Bristol Heart Institute, University of Bristol, Bristol, United Kingdom. 4. St. Louis University School of Medicine, St. Louis, Missouri, United States.


Lactate transport into and out of cells plays an important role in the distribution of energy-rich compounds within cells and tissues and is mediated mainly by different isoforms of monocarboxylate transporters (MCT). MCT isoforms 1-4 transport monocarboxylates such as lactate together with H+ in an electroneutral transport mode of 1:1 with different substrate affinities. We could recently show that carbonic anhydrase (CA) isoform II, comprised in a family of ubiquitous enzymes catalysing the equilibration of CO2, H+ and HCO3-, enhances transport activity of MCT1 and 4 when expressed in Xenopus oocytes (1, 2). The interaction between MCT1/4 and CAII did not depend on CA catalytic activity, but required the enzyme’s intramolecular H+ shuttle with residue His64 playing a central role (3, 4), suggesting that CAII might act as a “proton collecting antenna” for the carrier to ensure adequate rates of proton-lactate transport. We have now investigated possible interactions of the high-affinity MCT isoform 2 with cytosolic CAII as well as with extracellular CAIV, anchored to the membrane by a glycosylphosphatidylinositol (GPI) linkage. Activity of the proteins, heterologously expressed or injected in Xenopus laevis oocytes, was determined by measuring changes in intracellular H+ concentration with ion-selective microelectrodes in two-electrode voltage-clamp during application of lactate and CO2/HCO3-. Additionally, expression of MCT2 together with wild-type (WT) or mutants of CAIV was visualised by antibody staining, and possible changes in the level of protein expression were tested by Western blot analyses. Furthermore, catalytic activity of heterologously expressed CAs was determined by mass spectrometry. We could confirm former studies (5) that MCT2 needs embigin as ancillary protein for proper expression and catalytic function. In contrast to MCT1/4, activity of MCT2 was not affected by cytosolic CAII. However, extracellular CAIV-WT increased MCT2 transport activity, but only when embigin was coexpressed. The interaction between MCT2 and CAIV was persistent in the nominal absence of CO2/HCO3- as well as in the presence of the CA inhibitor ethoxyzolamide (EZA, 10 µM), indicating a non-catalytic facilitation of the transport activity. These findings were confirmed by coexpression of the catalytically inactive mutant CAIV-V165Y which also enhanced MCT2 transport activity. Moreover, coexpression of the mutant CAIV-H88A, in which a histidine, mediating an intramolecular H+ shuttle in CAIV, was exchanged, resulted only in a slight decrease in CAIV-mediated augmentation of MCT2 activity. The data suggest that extracellular, membrane bound CAIV augments transport activity of MCT2 in a non-catalytic manner, possibly by facilitating an alternative proton pathway. Supported by the DFG (GRK 845/3) and the Research Initiative Membrane Transport.

Where applicable, experiments conform with Society ethical requirements