Abstract
Excitatory amino acid transporters (EAATs) not only sustain a secondary active glutamate transport but also function as anion-selective ion channels. The relative proportion of currents generated by glutamate transport or by the chloride conductance varies for each cloned EAAT subtype. For EAAT1, EAAT2, and EAAT3, the anion current is only a small component of the total transporter-associated current amplitude, whereas EAAT4 and EAAT5 transporters mediate predominantly anion currents. We here demonstrate that the distinct current proportions are entirely due to differences in glutamate transport rates. EAAT3 and EAAT4 differ in unitary glutamate transport rates as well as in the voltage and substrate dependence of anion channel opening, but ion conduction properties are very similar. Noise analysis revealed identical unitary current amplitudes and similar absolute open probabilities for the two anion channels. The low glutamate transport rate of EAAT4 allows regulation of cellular excitability without interfering with extracellular glutamate homeostasis and makes this EAAT isoform ideally suited to regulate excitability in dendritic spines of Purkinje neurons.
Highlights
To obtain a better understanding of the functional basis of these differences, we compared the functional properties of two neuronal Excitatory amino acid transporters (EAATs) isoforms, EAAT3 and EAAT4
EAAT3 and EAAT4 anion channels differ in anion channel gating and glutamate dependence of the anion channel activation, two features that might be explained by a variable interaction between individual subunits of the multimeric transporter protein
Voltage-dependent Gating of EAAT3- and EAAT4-associated Anion Currents—We expressed hEAAT3 and rEAAT4 glutamate transporters heterologously in tsA201 cells and measured currents through whole cell patch clamp experiments
Summary
Heterologous Expression and Functional Characterization of EAAT3 and EAAT4—The coding region of hEAAT3 (GenBankTM accession number NP_004161) was excised from pTLN2-hEAAT3 Electrophysiology—Standard whole cell patch clamp recordings were performed using an Axopatch 200B (Molecular Devices, Sunnyvale, CA) amplifier as described [5, 8]. Glutamate uptake currents were measured by 100subtracting current amplitudes in a gluconate-based glutamate-free solution from values in an external solution supplemented with 500 M L-glutamate. Current-voltage relationships at various substrate concentrations were constructed by plotting isochronal current amplitudes determined 1 ms after the voltage step versus the membrane potential. 1 and 2) were determined by plotting the normalized isochronal current amplitude at ϩ135 or Ϫ135 mV after 0.2 s prepulses to different voltages versus the preceding potential. Isochronal anion current amplitudes were measured at various concentrations at a given test potential to obtain the concentration dependence of anion channel activation by glutamate. The substrate dependences obtained were normalized to the maximum current amplitude, averaged after normalization, and fit with the Hill equation
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