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Lucia Cadetti, Colette M. Maurer, Kerstin Dannenhauer, Ian C. Forster, Stephan C. Neuhauss; Charactherization of Zebrafish Excitatory Aminoacid Transporter EAAT2a and EAAT2b Expressed in Xenopus oocytes. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4310.
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© ARVO (1962-2015); The Authors (2016-present)
At the first retinal synapses, released glutamate is uploaded by EAATs located both on Muller cells and on photoreceptor terminals. Glutamate uptake is accompanied by the opening of an anion selective ion channel whose conductance has been shown to vary for different EAATs (Eliasof et al.1998; Torres-Salazar and Falke, 2007).In zebrafish, EAAT2a and EAAT2b are respectively expressed in Muller cells and photoreceptors (Maurer et al, in preparation). We analyze the characteristics of their associated anionic currents.
Xenopus laevis oocytes expressing either zebrafish EAAT2a or EAAT2b were studied by two-electrode voltage-clamp recording. Anionic currents were recorded in different extracellular solution and in presence of different L-glutamate concentrations. Radiolabeled glutamate uptake was performed with the aim of evaluating the charge to flux (charge transfer per molecule of uptaken glutamate) for each one of the two transporter.
L-Glutamate opened a conductance in oocytes expressing EAAT2a or EAAT2b. The L-glutamate-induced current was almost completely abolished once the oocytes were dialyzed for 24h in a chloride free solution and recorded in a gluconate based solution. This shows that, similar to EAAT mediated currents in other vertebrates, it was brought by Cl- ions. The current was induced by D-Aspartate as well and inverted at 21.00 + 8.28 mV for EAAT2a (N=5) and at 14.35 + 3.56 mV for EAAT2b (N=5). TBOA, an inhibitor of glutamate transporter, was more effective in blocking the chloride current in EAAT2a injected oocytes than in EAAT2b injected oocytes, where the block was often incomplete. TBOA (in absence of glutamate) induced an outward current at potentials below the chloride equilibrium potential in EAAT2b injected oocytes while it had no effect on EAAT2a injected oocytes. Finally, the charge to flux rate was determined for both EAATs and it was shown to be about 3 times larger for EAAT2b (N=11) than for EAAT2a (N=8).
For both EAAT2a and 2b, currents elicited by L-glutamate are almost entirely constituted by a flux of Cl- ions. The Cl- conductance is 3 times bigger in EAAT2b than in EAAT2a, in agreement with suggested EAAT function in retinal physiology: EAAT2a is indeed expressed in zebrafish Muller cells and its main function is uptake of glutamate to prevent excitotoxicity and spillover, while EAAT2a, through a larger Cl- conductance, play an important role in photoreceptor feedback (Veruki et al,2006; Szmajda and Devries, 2011) and therefore in retinal signal processing.
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