Abstract
Glutamate is the major excitatory neurotransmitter, but prolonged exposure even at micromolar concentrations causes neuronal death. Extracellular glutamate is maintained at nanomolar level by glutamate transporters, which, however, may reverse transport and release glutamate. If and when the reverse occurs depends on glutamate transport stoichiometry (GTS). Previously we found that in the presence of chloride, the coupled GLT-1 glutamate transporter current and its relationship to radiolabeled glutamate flux significantly decreased when extracellular glutamate concentration increased above 0.2 mM, which implies a change in GTS. Such high concentrations are feasible near GLT-1 expressed close to synaptic release site during excitatory neurotransmission. The aim of this study was to determine GLT-1 GTS at both low (19–75 μM) and high (300–1200 μM) glutamate concentration ranges. GTS experiments were conducted in the absence of chloride to avoid contributions by the GLT-1 uncoupled chloride conductance. Mathematical analysis of the transporter thermodynamic equilibrium allowed us to derive equations revealing the number of a particular type of ion transported per elementary charge based on the measurements of the transporter reversal potential. We found that GLT-1a expressed in COS-7 cells co-transports 1.5 Na+, 0.5 Glu-, 0.5 H+ and counter-transports 0.6 K+ per elementary charge in both glutamate concentration ranges, and at both 37°C and 26°C temperatures. The thermodynamic parameter Q 10 = 2.4 for GLT-1 turnover rate of 19 s-1 (37°C, -50 mV) remained constant in the 10 μM–10 mM glutamate concentration range. Importantly, the previously reported decrease in the current/flux ratio at high glutamate concentration was not seen in the absence of chloride in both COS-7 cells and cultured rat neurons. Therefore, only in the absence of chloride, GLT-1 GTS remains constant at all glutamate concentrations. Possible explanations for why apparent GTS might vary in the presence of chloride are discussed.
Highlights
Glutamate is the major excitatory neurotransmitter of the central nervous system, but exposures to even low glutamate concentrations are toxic to neurons [1,2]
GLT-1 glutamate transport stoichiometry (GTS) is constant with substitution of gluconate for chloride
Analysis of GLT-1 glutamate transport thermodynamic equilibrium allowed us to derive Eqs (7–10, see the methods) showing that the number of a particular type of ion transported per elementary charge is equal to the ratio of the transporter reversal potential shift to the particular ion’s Nernst equilibrium potential shift
Summary
Glutamate is the major excitatory neurotransmitter of the central nervous system, but exposures to even low glutamate concentrations are toxic to neurons [1,2]. The GTS of glutamate transporters EAAT3, GLT-1 and GLAST has been determined previously as 3Na+: 1Glu-: 1H+: (-1)K+ per transport cycle [6,7,8]. All these published studies have been performed in the absence of chloride to avoid contaminating the measurement of the transporter current by the uncoupled chloride current, which is transmitted via an anionic channel associated with glutamate transporters [9,10]. The possibility of GTS variation with changes in glutamate concentration has never been explored
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