Abstract
Excitatory amino acid transporters (EAATs) mediate two distinct transport processes, a stoichiometrically coupled transport of glutamate, Na+, K+, and H+, and a pore-mediated anion conductance. We studied the anion conductance associated with two mammalian EAAT isoforms, hEAAT2 and rEAAT4, using whole-cell patch clamp recording on transfected mammalian cells. Both isoforms exhibited constitutively active, multiply occupied anion pores that were functionally modified by various steps of the Glu/Na+/H+/K+ transport cycle. Permeability and conductivity ratios were distinct for cells dialyzed with Na(+)- or K(+)-based internal solution, and application of external glutamate altered anion permeability ratios and the concentration dependence of the anion influx. EAAT4 but not EAAT2 anion channels displayed voltage-dependent gating that was modified by glutamate. These results are incompatible with the notion that glutamate only increases the open probability of the anion pore associated with glutamate transporters and demonstrate unique gating mechanisms of EAAT-associated anion channels.
Highlights
Excitatory amino acid transporters (EAATs)1 mediate the removal of glutamate from the synaptic cleft in the central nervous system and the uptake of glutamate in kidney and intestine [1,2,3]
EAAT-associated Anion Currents Are Active in the Absence of Transporter Substrate—Fig. 1 shows representative recordings from tsA201 cells expressing EAAT2 and EAAT4
Application of external glutamate caused a 2-fold increase of the anion current amplitude (Fig. 4A) indicating that EAAT-associated anion channels are active in the absence of substrate and that this tonically active anion conductance is a substantial component of the activated anion current (Figs. 3 and 4)
Summary
Expression of hEAAT2 and rEAAT4 in tsA201 and HEK293 Cells—A pcDNA3.1-hEAAT2 construct was generated by subcloning the coding region of hEAAT2 [16] The subtracted variances were sorted into current bins [25] and plotted versus the corresponding mean current amplitude (Fig. 6) Under these experimental conditions the absolute open probability changes with time as given by Equation 2, yielding Equation 3. A fit of Equation 3 to the variance versus mean current [24] plot allowed us to determine the unitary current amplitude and the number of anion channels By using these two values we calculated the absolute open probabilities (P) at the holding potential by extrapolating the instantaneous macroscopic current amplitude (Iinst) and dividing this amplitude by the number of channels (N) and the single channel amplitude (i) as shown in Equation 4
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