Sulfhydryl modification of V449C in the glutamate transporter EAAT1 abolishes substrate transport but not the substrate-gated anion conductance

Abstract

Excitatory amino acid transporters (EAATs) buffer and remove synaptically released L-glutamate and maintain its concentrations below neurotoxic levels. EAATs also mediate a thermodynamically uncoupled substrate-gated anion conductance that may modulate cell excitability. Here, we demonstrate that modification of a cysteine substituted within a C-terminal domain of EAAT1 abolishes transport in both the forward and reverse directions without affecting activation of the anion conductance. EC(50)s for L-glutamate and sodium are significantly lower after modification, consistent with kinetic models of the transport cycle that link anion channel gating to an early step in substrate translocation. Also, decreasing the pH from 7.5 to 6.5 decreases the EC(50) for L-glutamate to activate the anion conductance, without affecting the EC(50) for the entire transport cycle. These findings demonstrate for the first time a structural separation of transport and the uncoupled anion flux. Moreover, they shed light on some controversial aspects of the EAAT transport cycle, including the kinetics of proton binding and anion conductance activation.

Figure 1

Effect of MTSET on Cysless and V449C transporters. (A) Application of 100 μM l-glutamate (open bar) generates a steady-state current in oocytes expressing the V449C carrier (Vm = −60 mV). After a 4-min application of 1 mM MTSET (shaded bar) and a 2-min washout, a second application of 100 μM l-glutamate (open bar) generates a steady-state current of smaller amplitude. (B) The same protocol carried out on the Cysless transporter results in steady-state currents of similar amplitude.

Figure 2

Effect of MTS derivatives on the I–V relationships generated by the V449C, Cysless, and EAAT1 transporters. Voltage jumps were made both before (open squares) and after (filled squares) application of 1 mM MTSET (2 min) to oocytes expressing the V449C (A), Cysless (B), or EAAT1 (C) transporters. Control currents were subtracted from currents generated in the presence of 100 μM l-glutamate.

Figure 3

Sodium dependence of the steady-state currents generated by V449C. I–Vs generated by the application of 100 μM l-glutamate before (open symbols) and after (filled symbols) application of 1 mM MTSET and in the presence of extracellular sodium (squares) or choline (circles). Control currents were subtracted from currents generated in the presence of 100 μM l-glutamate.

Figure 4

Characterization of the anion conductance generated by the V449C carrier. (A) The reversal potential of the l-glutamate-elicited currents generated by the modified V449C transporter was measured at three concentrations of extracellular chloride (103.6, 55.6, and 7.6 mM). A 10-fold change in the extracellular chloride concentration resulted in a 58 ± 3 mV shift in the reversal potential. Each point is the average ± SEM from ten oocytes. (B) Extracellular anion substitutions. I–Vs elicited by 100 μM l-glutamate were measured with chloride (squares), bromide (triangles), or nitrate (circles) as the extracellular anion both before (closed symbols) and after (open symbols) modification by MTSET. Control currents were subtracted from currents generated in the presence of 100 μM l-glutamate.

Figure 5

Radiolabeled uptake by the V449C and Cysless transporters held under voltage clamp. Oocytes were voltage clamped (Vm = −60mV) and 100 μM d-[³H]aspartate (shaded squares) or l-[³H]glutamate (open squares) was applied for 4 min followed by a 2-min washout. Each value is the average ± SEM for n ≥ 4.

Figure 6

dl-TBOA-sensitive currents mediated by V449C and Cysless during reverse transport before and after modification. I–V curves elicited by 40 mM potassium nitrate (60 mM sodium nitrate) were measured from V449C- or Cysless-expressing or uninjected oocytes. I–Vs were subtracted from those measured in the presence of 100 μM dl-TBOA. Before modification, the I–V for V449C (A, open squares) was similar to Cysless (B, open squares). After application of 1 mM MTSET, the I–V for V449C (A, filled squares) was similar to uninjected oocytes (C).