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Ion Fluxes Associated with Excitatory Amino Acid Transport

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Ion Fluxes Associated with Excitatory Amino Acid Transport Neuron, Vol. 15, 721-728, September, 1995, Copyright © 1995 by Cell Press Ion Fluxes Associated with Excitatory Amino Acid Transport Jacques I. Wadiche,* Susan G. Amara,t et al., 1992). Three homologous excitatory amino acid and Michael P. Kavanaugh* transporter (EAAT) subtypes are widely expressed in hu- *Vollum Institute man brain (EAAT1, EAAT2, and EAAT3; Arriza et al., tHoward Hughes Medical Institute 1994). A kinetic study of one of the human transporters Oregon Health Sciences University (EAAT2; Wadiche et al., 1995) demonstrated that the num- Portland, Oregon 97201 ber of charges translocated per transport cycle varies ac- cording to the membrane potential, in contrast to the result expected for a simple transport model involving a fixed Summary stoichiometry. Data are presented here that explain the basis for this variable stoichiometry by demonstrating that Flux of substrate and charge mediated by three cloned members of this transporter family mediate thermodynam- excitatory amino acid transporters widely expressed ically uncoupled CI- currents activated by the molecules in human brain were studied in voltage-clamped Xeno- they transport. pus oocytes. Superfusion of L-glutamate or D-aspartate resulted in currents due in part to electrogenic Na ÷ Results cotransport, which contributed 1 net positive charge per transport cycle. A significant additional compo- Steady-State Currents Activated by Excitatory nent of the currents was due to activation of a revers- Amino Acids ible anion flux that was not thermodynamically coupled The voltage dependence of the currents mediated by to amino acid transport. The selectivity sequence EAAT1, EAAT2, and EAAT3 was examined by clamping of this ligand-activated conductance was NO3- > I- > oocytes expressing the transporters at potentials between Br > CI- > F-. The results suggest that these proteins +60 and -30 mV and superfusing them with the transport mediate both transporter- and channel-like modes of substrate D-aspartate (Arriza et al., 1994) at 100 ~M. At permeation, providing a potential mechanism for negative membrane potentials, amino acid superfusion dampening cell excitability, in addition to removal of induced inward currents in oocytes expressing all three transmitter. transporter subtypes (Figures tA, 1C, and 1E). The amino acid-dependent current mediated by EAAT2 did not re- Introduction verse at potentials up to +60 mV (Figures 1C and 1D). Surprisingly, however, amino acid superfusion induced Reuptake of neurotransmitters is mediated by specific currents that reversed at positive membrane potentials in membrane proteins that couple the electrochemical gradi- oocytes expressing EAAT1 (Erev = 9.3 _.+ 0.7 mV; n -- ents of additional cotransported ions to drive the concen- 46; Figures 1A and 1 B) or EAAT3 (Erov - 38.0 __. 2.7 mV; trative influx of transmitter (for review, see Lester et al., n = 28; Figures 1E and 1F). The outward currents were 1994). The molecular mechanisms underlying this cou- most likely not due to reverse transport of accumulated pling process are unknown. Voltage-clamp studies have substrate because they were observed in response to the revealed substrate-independent ion fluxes mediated by first application of amino acid, when the membrane was some cloned neurotransmitter transporters, including those clamped at depolarized potentials (Figure 1). In addition, for 5-hydroxytryptamine (Mager et al., 1994), ~(-aminobutyric varying external concentrations of amino acid affected the acid (Cammack et al., 1994), and glutamate (Vandenberg amplitudes of the currents without changing the reversal et al., 1995), suggesting the presence of channel-like prop- potential (Figure 2). In the absence of Na÷ (choline substi- erties in these molecules. Uptake of the excitatory amino tution), neither inward nor outward currents were induced acid neurotransmitters glutamate and aspartate in brain by amino acid superfusion in any of the three transporter synaptosomes is associated with influx of Na+ and effiux subtypes (n = 4). The application of 1 mM L-glutamate of K ÷ and OH- (Kanner and Sharon, 1978; Erecinska et al., or D-aspartate to water-injected oocytes did not activate 1983). Voltage-clamp recording in retinal photoreceptors a detectable current (n = 6). and glial cells has been used to isolate currents associated with excitatory amino acid uptake that contribute signifi- A Component of the Transporter Current cantly to these cells' electrical properties (Brew and Att- Is Carried by CI- well, 1987; Tachibana and Kaneko, 1988; Schwartz and The amino acid-dependent outward current observed at Tachibana, 1990; Eliasof and Werblin, 1993). A stoichiom- positive potentials in oocytes expressing EAAT1 or EAAT3 etry proposed for glutamate uptake involves cotransport was abolished when external CI- ions (CI-out)were replaced of 2Na+:lGlu - with countertransport of 1 K + and 1 OH-, by gluconate ions, with little effect on the inward currents resulting in translocation of I net positive charge (Bouvier (Figures 3A-3C). This result suggested the possibility that et al., 1992). the outward currents mediated by EAAT1 and EAAT3 were Isolation of cDNA clones from rat and rabbit has re- carried by CI-. This was tested by examination of the rever- vealed a mammalian gene family of glutamate transport- sal potential of the amino acid-dependentcurrents medi- ers (Kanai and Hediger, 1992; Pines et al., 1992; Storck ated by EAAT1 and EAAT3 as a function of the extracellu- Neuron 722 A EAAT 1 C EAAT2 E EAAT 3 L +60 mV +60 mV ) tooo, a¢ 0 see -30 mV B +300 D F +200 +100 +20 +100 membrane potential (mV) membrane potential (mV) -40 -20 _ +20 +40 w+6~O +80 -40 -20 +20 +40 • • +80 P-- I • 9 ---t--- I I I-- I I I-- I -40 -20 +20 +40 +60 +50 membrane potential (mV) -20 -100 • -200 -200 -300 -60 • -300 o= -80 -400 Figure 1. Currents Mediated by Human Excitatory Amino Acid Transporters Currents induced by bath application (indicated by bar) of 100 pM D-aspartate to Xenopus oocytes expressing EAAT1 (A), EAAT2 (C), and EAAT3 (E). The corresponding steady-state current-voltage relations are shown in (B), (D), and (F). Currents are recorded at potentials from -30 to +60 mV and are offset to align holding currents. Note outward currents observed for EAAT1 and EAAT3. lar CI- concentration ([CI-]o°,). The reversal potential of the tion of the transporter-mediated current activated by excit- EAAT1 and EAAT3 currents shifted 54.1 ± 1.8 mV (n = atory amino acids. 5) and 53.7 ± 4.3 mV (n = 5) per decade change in [CI-]out, To examine the properties of the transporter currents respectively (Figure 3D). Although the magnitudes of the further, intracellular CI- (Cl-~o) was depleted by dialysis of reversal potential shifts were close to predictions for a EAATl-expressing oocytes for 16-24 hr at 17°C in CI--free CI--selective electrode, the absolute values of the reversal medium (gluconate substitution); these oocytes were com- potentials were significantly more positive than the value pared with matched oocytes incubated in ND96. Ec~ was of Ec~, the CI- equilibrium potential (see below). This result measured before and after CI-in depletion by measurement suggested that other ions in addition to CI carried a por- of reversals of endogenous Ca2%dependent CI- channels A EAAT 1 B EAAT 2 C EAAT 3 membrane potential (mV) +300 membrane potential (mV) +50 membrane potential (mV) I +500 -150 -100 -50 -t O -,00 -50 +100 -150 -1SO -50 J ~ +100 o a o o o eo~e o~o~o -300 o o ~ ~ -soo e oq°° o ~o ~ o A A <>~ o uG -600 " -100 A o~ ¢ o <> Do -1000 o~ Ao~ o oo -900 ~ o Ao y -150 ~" u o -1500 AoU El o o 0 o El -1200 o ~ -200 o L] o -2000 o -1500 -250 -2500 Figure 2. Concentration and Voltage Dependence of Excitatory Amino Acid Currents Steady-state current-voltage relations of representative oocytes between +80 and -120 mV, obtained by subtraction of control currents from the corresponding currents in the presence of varying concentrations of D-aspartate. Xenopus oocytes expressing EAAT1 (A), EAAT2 (B), and EAAT3 (C). The reversal potential (EAAT1 and EAAT3) does not shift as a function of [D-aspartate]. D-aspartate doses were 10 I~M (closed circles), 30 p.M (triangles), 100 ~M (diamonds), 300 p.M (squares), and 1 mM (open circles). Excitatory Amino Acid Transporter Ion Ftuxes 723 A B C EAAT 1 EAAT 2 EAAT 3 +s00 104raM Cl~= .so 104mM Cir.t 104mM Cl~ut membrane potential (mV) ~o membrane potential (mV) mernbrane potential (rnV) ~[ +500 ~o,// +tnn -150 -100 -,50 ot]~o° +100 -150 -100 -50 /.00 -150 -100 -50 I L ~ ~-~o-b~d I I I __ L i 1 ~e o 9 I ~o ,I°B~[3oD~O ~o \ ~o -500 [3o -soo 0mM Cl~ut -so OmM C/-= ea ss' OmM CI~, ~o 0 -1000 -,o00 o= -100 2 o ~: -1500 -1500 ~' g -100 = © = u g -2000 ~'> -2000 E] -200 -2500 D Figure 3, CI- Carries the Transporter-MediatedOutward Current (A-C) Steady-statecurrent-voltage relationsof oocytes expressing 80 EAAT1 (A), EAAT2 (B), and EAAT3 (C) in responseto an application 70 of 1 mM D-aspartate.Removal of CI oct (gluconatesubstitution) abol- ishes the outward current but has no effect on the inward current. 60 (D) Reversalpotential of the current inducedby D-aspartateis depen- 5o dent on [CI ]o°t.The reversalsshifted 54.1 _+ 1.8 and 53.7 + 4.3 mV/ ~ 40 decade for EAAT1 and EAAT3, respectively(mean _+ SEM; n = 5), uJ~ 30 20 10 0 10 100 [CI-]out (mi) (see Experimental Procedures). With 104 mM [CI-]out, Ec~ dent current-voltage relations (n = 5). In addition, the was shifted from -17 - 1 mV (n = 4) to -81 _ 3 mV oocyte CI- channel blocker niflumic acid (100 I~M; White (n = 3), avalue corresponding to a change in intracellular and Aylwin, 1990) and the nonselective blocker SITS (1 concentration from 53 to 4 raM.
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