Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCA098

Poster Communications

A proton antenna in carbonic anhydrase II supports lactate transport in cancer cells

S. I. Noor1, S. Jamali1, S. Ames1, J. W. Deitmer1, H. M. Becker1,2

1. Division of General Zoology, Department of Biology, University of Kaiserslautern, Kaiserslautern, Germany. 2. Institute of Physiological Chemistry, University of Veterinary Medicine Hannovericine Hannover, Hannover, Germany.


  • Figure 1: CAII functions as H+ antenna for MCTs. Intracellular CAII is anchored to MCT1/4 via CAII-His64. This binding brings CAII close enough to the MCT to shuttle H+ between transporter and surrounding protonatable residues. H+ transfer is mediated by CAII-Glu69 and CAII-Asp72. Under hypoxic conditions CAIX could bind to MCT1/4 via their chaperon CD147 to facilitate the exchange of H+ between transporter and extracellular protonatable residues in a similar fashion as CAII [2, 3]. By this non-catalytic mechanism intracellular and extracellular carbonic anhydrases could cooperate non-enzymatically to facilitate H+-driven lactate flux across the cell membrane of cancer cells.

Tumor cells, especially those that reside in a hypoxic environment, produce vast amounts of lactate and acid, which have to be quickly removed from the cell to avoid intracellular lactacidosis and suffocation of metabolism. Lactate efflux from cancer cells is mediated by H+-coupled monocarboxylate transporters (MCTs). In the present study, we investigated whether lactate flux in MCF-7 breast cancer cells is facilitated by the intracellular carbonic anhydrase CAII. Knockdown of CAII, but not inhibition of CA catalytic activity with EZA, reduced H+-coupled lactate transport in normoxic and hypoxic MCF-7 cells, as determined by measuring changes in intracellular pH with the fluorescent dye SNARF-5F. Furthermore, knockdown of CAII resulted in a significant reduction in cell proliferation, as did inhibition of lactate transport with AR-C155858, while inhibition of CA catalytic activity with EZA did not influence proliferation. An in situ proximity ligation assay demonstrated that CAII is closely colocalized to MCT1, which suggests a direct interaction between the proteins. The mechanism behind this interaction was elucidated by coexpression of MCT1 and MCT4 with various CAII mutants in Xenopus oocytes. CAII facilitates MCT transport activity via CAII-Glu69 and CAII-Asp72, which have been suggested to function as surface H+ antenna for the enzyme [1]. However, determination of CAII catalytic activity with gas-analysis mass spectrometry revealed that Glu69 and Asp72 are not involved in catalytic function. While CAII-Glu69 and CAII-Asp72 seem to mediate H+ transfer between enzyme and transporter, CAII-His64, the central residue of the enzyme's intramolecular H+ shuttle, is not involved in H+ transfer between the two proteins, but mediates binding of CAII to the transporter's C-terminal tail, as shown by a pull-down assay with GST-fusion proteins. Taken together, the results suggest that CAII features a moiety that exclusively mediates H+ exchange with the transporter, while the central residue of the intramolecular H+ shuttle, His64, mediates binding to MCT. Thereby CAII drives the MCT-mediated efflux of H+ and lactate from the cell to support cellular metabolism and proliferation (Fig. 1).

Where applicable, experiments conform with Society ethical requirements