Glutamate Modifies Ion Conduction and Voltage-dependent Gating of Excitatory Amino Acid Transporter-associated Anion Channels

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. 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 (1-3). Five structurally distinct subtypes of mammalian glutamate transporters, EAAT1-EAAT5, have been identified in recent years (4 -9). Each of these isoforms exhibits two separate transport processes: a stoichiometrically coupled cotransport of one glutamate with three Na ϩ ions and one H ϩ , in countertransport to one K ϩ ion (10, 11); and an anion conductance that appears to be pore-mediated. The EAAT-associated anion channel is currently thought to function as a glutamategated channel with a tight coupling of channel opening and closing to conformational changes of the corresponding carrier domain. In this model, only certain carrier conformations are associated with conducting anion pores, and the anion channel cycles between conducting and non-conducting states during transitions through various conformational states of the glutamate carrier (8,9,(12)(13)(14)(15).We investigated anion conduction properties of two EAAT isoforms, human EAAT2 and rat EAAT4, using patch clamp recordings of transfected tsA201 cells under conditions that eliminate the Glu/Na ϩ /H ϩ /K ϩ current component. Our results demonstrated that opening and closing of the EAAT-associated anion channels as well as the interaction with the glutamate uptake process are more complex than previously assumed. External glutamate modifies intrinsic properties of EAAT2-and EAAT4-associated anion channels such as anion selectivity and the rate constants of anion permeation. Moreover, EAAT4 anion channels exhibit voltage-dependent opening and closing transitions that are modified by glutamate. These results provide novel insights into the function of neurotransmitter transporters and illustrate similarities as well as differences between transporter-associated pores and ion channe...

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“…For all tested hEAAT2 constructs, a comparable constitutive anion current was observed in the absence of glutamate (Fig. 1B) that was about 2-fold increased by external glutamate (12). We conclude that the addition of the His tag does not alter the coupled and the uncoupled current amplitudes of hEAAT2.…”

“…1A). In a SCN Ϫ -based external solution, hEAAT2 exhibited anion currents that largely exceeded the Glu/Na ϩ /H ϩ /K ϩ current component allowing to directly measure anion currents (12). For all tested hEAAT2 constructs, a comparable constitutive anion current was observed in the absence of glutamate (Fig.…”

“…The standard external solution contained 96 mM NaCl, 4 mM KCl, 0.3 mM CaCl 2 , 1 mM MgCl 2 , 5 mM HEPES, pH 7.4, and the glutamatecontaining solution was supplied with 0.5 mM L-glutamate. Anion currents were determined after exchanging the external solution to 96 mM NaSCN, 4 mM KCl, 0.3 mM CaCl 2 , 1 mM MgCl 2 , 5 mM HEPES, pH 7.4, in the absence and presence of 0.5 mM external glutamate without any current subtraction procedure (12). Anion currents were normalized by dividing current amplitudes by the Glu/Na ϩ /H ϩ /K ϩ uptake current amplitude measured at Ϫ140 mV.…”

“…Anion currents were normalized by dividing current amplitudes by the Glu/Na ϩ /H ϩ /K ϩ uptake current amplitude measured at Ϫ140 mV. Glutamate uptake currents in mammalian cells were measured through standard whole-cell patch clamp recordings using an Axopatch 200B (Axon Instruments) amplifier as described previously (12). Currents were filtered at 5 kHz and digitized with a sampling rate of 50 kHz using a Digidata (Axon Instruments).…”

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“…34 In addition to mediating secondary-active glutamate uptake, EAATs also function as anion channels. [35][36][37][38] The EAAT1 p.Pro290Arg variant decelerates a conformational change associated with Na þ binding, causing a decrease in glutamate transport rates and a significant increase in EAAT1-associated anion current amplitudes. 39 EAAT1 and EAAT2 have similar expression profiles (mainly expressed in astrocytes and localized to plasmalemma), share basic mechanisms of glutamate transport, and exhibit similar anion conduction properties.…”