Arsenite-treated Red Blood Cells: transport or binding?

Re: "Chemical activation of a high-affinity glutamate transporter in human erythrocytes and its implications for malaria-parasite–induced glutamate uptake" Winterberg, et al., 119:3604-3612doi:10.1182/blood-2011-10-386003 Winterberg et al. (Blood 2012; 119: 3604-3612) report that the treatment of human erythrocytes with arsenite or their infection with P. falciparum induces a transport system for glutamate with the characteristics of the "excitatory amino acid transporter" (EAAT) family and likely corresponding to the EAAT3 transporter. Authors present functional results and expression data from immunofluorescence and Western Blot experiments. Western Blot was performed with a monoclonal anti-EAAT3 More...Winterberg et al. (Blood 2012; 119: 3604-3612) report that the treatment of human erythrocytes with arsenite or their infection with P. falciparum induces a transport system for glutamate with the characteristics of the "excitatory amino acid transporter" (EAAT) family and likely corresponding to the EAAT3 transporter. Authors present functional results and expression data from immunofluorescence and Western Blot experiments. Western Blot was performed with a monoclonal anti-EAAT3 antibody in uninfected, untreated RBCs and immunofluorescence in control and infected RBCs but no data are reported on arsenite-treated RBCs. We question Authors' conclusions for arsenite-treated RBCs at the light of these arguments: a) EAAT transporters are highly concentrative, having a stoichiometry of 3 Na+ + 1 H+ co-transported and 1 K+ counter-transported for each amino acid amino acid (1). Yet, no uphill accumulation of substrates is observed in arsenite-treated cells or in P. falcifarum infected erythrocytes (see Figure 1). As far as infected RBCs are concerned, Authors attribute the low gradient to their high sodium content, but what about RBCs treated with arsenite? b) Due to the stoichiometry of EAAT transporters, the dependence of substrate influx on sodium should be sigmoidal (Hill coefficient = 2 or even higher (2)). Yet, the dependence on sodium of glutamate influx in arsenite-treated cells is hyperbolic (see Fig. 3), strongly suggesting a 1:1 stoichiometry. c) The Km reported for glutamate transport in arsenite-treated cells is 55 +/- 9 nM, that is 200-fold lower than the Km value generally observed for EAAT3 (1). Finally, we wonder what can be the physiological meaning of the findings reported by Winterberg et al., given that the Vmax values they obtain would indicate that less than 200 molecules of glutamate are transported in each RBC per minute. We think that it cannot be excluded that arsenite-treated human RBCs express a glutamate transporter exhibiting novel features, rather than those typical of EAAT3, or that, alternatively, Winterberg et al. measure binding rather than true transport. Massimiliano G. Bianchi, Ph.D., and Ovidio Bussolati, M.D. Ph.D. Unit of General Pathology Department of Biomedical, Biotechnological, and Translational Sciences University of Parma I-43125, Parma, Italy 1) Kanai Y. and Hediger M.A. The glutamate/neutral amino acid transporter family SLC1: molecular, physiological and pharmacological aspects. Pflugers Arch 2004; 447: 469-479. 2) Kanai Y., Nussberger S., Romero M.F., Boron W.F., Hebert S.C. and Hediger M.A. Electrogenic properties of the epithelial and neuronal high affinity glutamate transporter. J Biol Chem; 1995; 270: 16561-16568.