Anion Conductance Behavior of the Glutamate Uptake Carrier in Salamander Retinal Glial Cells

Abstract

Glutamate uptake is driven by the cotransport of Na+ ions, the countertransport of K+ ions, and either the countertransport of OH- or the cotransport of H+ ions. In addition, activating glutamate uptake carriers has been shown to lead to activation of an anion conductance present in the carrier structure. Here we characterize the ion selectivity and gating of this anion conductance. The conductance is small with Cl- as the permeant anion, but it is large with NO3- or ClO4- present, undermining the earlier use of NO3- and ClO4- to suggest that OH- countertransport rather than H+ cotransport helps drive uptake. Activation of the anion conductance can be evoked by extra- or intracellular glutamate and can occur even when glutamate transport is inhibited. By running the carrier backward and detecting glutamate release with AMPA receptors in neurons placed near the glial cells, we show that anion flux is not coupled thermodynamically to glutamate movement, but OH-/H+ transport is. The possibility that cell excitability is modulated by the anion conductance associated with glutamate uptake suggests a target for therapeutic drugs to reduce glutamate release in conditions like epilepsy.

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

“…Most kinetic models of the transport cycle describe the existence of an anion-conducting state after the substrate and cotransported ions have bound to the extracellular side of the transporter and before their translocation (14)(15)(16)(17)(18). In an alternative model the anion conducting state has been proposed to follow the translocation of substrate and cotransported ions (19). Our finding that modification impairs substrate translocation but not the anion conductance supports the existence of an anionconducting state before translocation.…”

“…In both heterologous and native systems expressing EAATs, reversal of the transmembrane electrochemical gradients for sodium and potassium ions leads to an outward flux of intracellular glutamate (3) and the activation of the transporterassociated anion conductance (19,21). To determine whether the modified carrier can catalyze reverse transport and͞or activate the anion conductance when ionic gradients are altered to favor substrate efflux, we applied a high potassium (40 mM), low sodium (60 mM) buffer to oocytes expressing the V449C or Cysless carriers.…”

“…Studies using patch clamp analyses to measure rapid transport kinetics have linked gating of the anion conductance to early steps in the transport cycle (14)(15)(16)(17)(18). Association of anion channel gating events with the translocation limb of the cycle is further supported by the observation that transporters locked in an exchange mode still mediate the anion conductance (19)(20)(21)(22)(23). However, because substrates activate the anion channel activity at membrane potentials (Ͼ70 mV) and temperatures (Ͻ4°C) that greatly restrict transport (14), translocation may not be required.…”

Glutamate transport by retinal Müller cells in glutamate/aspartate transporter‐knockout mice

“…It has been demonstrated that in addition to the uptake of glutamate, EAATs are coupled to an anion conductance that allows for larger currents when anions other than Cl Ϫ are used (Eliasof and Jahr, 1996;Billups et al, 1996). Therefore, we examined Mü ller cell responses to 1mM glutamate when extracellular Cl Ϫ was replaced by SCN Ϫ .…”

Cellular Neurophysiology

“…Since glutamate uptake is increased when pHi is more alkaline (Billups & Attwell, 1996), we investigated whether the potentiation of uptake by NHs was due to a change of pHi' Raising the Hepes concentration in the pipette from 5 to 71 mM, to clamp pHj at 7'0 more effectively, did not significantly alter the potentiation of the glutamate-evoked current (21 ± 2 %, n = 27). Potentiation of the glutamate-evoked current was smaller (7 ± 2 %, n = 19) but not abolished when the pH of the pipette solution was raised to 8'8 (a value at which the uptake current is not increased by further alkalinization; Billups & Attwell, 1996).…”