Anion Conducting States of Excitatory Amino Acid Transporters

Excitatory amino acid transporters (EAATs) are secondary active, electrogenic transporters which translocate L-glutamate (glu) against its concentration gradient using the co-transport of 3 Na+, 1 H+, and the counter-transport of 1 K+ ion. In addition, these carriers possess a thermodynamically uncoupled anion channel that fluxes Cl- but is promiscuous with several permeant anionic species. The roles of EAATs are to shape the spatio-temporal profile of released glu in both the synaptic cleft and extra-synaptic regions as well as maintaining a low ambient extracellular concentration of glu. This transport activity regulates activation of glu receptors and thus regulates excitatory neurotransmission. Using a combination of techniques, we were successful in identifying inward oriented transporter conformations which allow transitions to open channels states. This observation was enabled by our development of a novel method to isolate EAAT1 in the inward facing conformation. While constrained to these conformations, currents with the same macroscopic amplitudes as conducting states mediated by the outward facing, Na+ bound states were observed. The persistence of currents is indicative of a channel gating mechanism that is insensitive to transporter orientation and that the anion channel is open during the majority of the transport cycle. Additional conducting states allows for a larger contribution of the anion channel function of EAATs to shape cellular function then previously assumed. Next we investigated the gating mechanism of the anion channel. We assayed for the ability of Na+ to gate the anion channel in both glial (EAAT1 and EAAT2) and neuronal (EAAT3 and EAAT4) isoforms. We discovered that the glial isoforms are not gated by Na+ but are leak channels with an open probability and single channel conductance that is insensitive to Na+ concentrations. In contrast, neuronal EAAT isoforms EAAT3 and EAAT4 both display Na+ dependent channel activity. This is the first example of a significant functional difference between glial and neuronal transporter isoforms of the solute carrier 1 (SLC1) family. The research presented here allows for a greater understanding of low open probability channel states and the possible contributions of the EAAT anion channel to the functioning of the nervous system

Loss of VGLUT3 Produces Circadian-Dependent Hyperdopaminergia and Ameliorates Motor Dysfunction and l-Dopa-Mediated Dyskinesias in a Model of Parkinson\u27s Disease.

UNLABELLED: The striatum is essential for many aspects of mammalian behavior, including motivation and movement, and is dysfunctional in motor disorders such as Parkinson\u27s disease. The vesicular glutamate transporter 3 (VGLUT3) is expressed by striatal cholinergic interneurons (CINs) and is thus well positioned to regulate dopamine (DA) signaling and locomotor activity, a canonical measure of basal ganglia output. We now report that VGLUT3 knock-out (KO) mice show circadian-dependent hyperlocomotor activity that is restricted to the waking cycle and is due to an increase in striatal DA synthesis, packaging, and release. Using a conditional VGLUT3 KO mouse, we show that deletion of the transporter from CINs, surprisingly, does not alter evoked DA release in the dorsal striatum or baseline locomotor activity. The mice do, however, display changes in rearing behavior and sensorimotor gating. Elevation of DA release in the global KO raised the possibility that motor deficits in a Parkinson\u27s disease model would be reduced. Remarkably, after a partial 6-hydroxydopamine (6-OHDA)-mediated DA depletion (∼70% in dorsal striatum), KO mice, in contrast to WT mice, showed normal motor behavior across the entire circadian cycle. l-3,4-dihydroxyphenylalanine-mediated dyskinesias were also significantly attenuated. These findings thus point to new mechanisms to regulate basal ganglia function and potentially treat Parkinson\u27s disease and related disorders. SIGNIFICANCE STATEMENT: Dopaminergic signaling is critical for both motor and cognitive functions in the mammalian nervous system. Impairments, such as those found in Parkinson\u27s disease patients, can lead to severe motor deficits. Vesicular glutamate transporter 3 (VGLUT3) loads glutamate into secretory vesicles for neurotransmission and is expressed by discrete neuron populations throughout the nervous system. Here, we report that the absence of VGLUT3 in mice leads to an upregulation of the midbrain dopamine system. Remarkably, in a Parkinson\u27s disease model, the mice show normal motor behavior. They also show fewer abnormal motor behaviors (dyskinesias) in response to l-3,4-dihydroxyphenylalanine, the principal treatment for Parkinson\u27s disease. The work thus suggests new avenues for the development of novel treatment strategies for Parkinson\u27s disease and potentially other basal-ganglia-related disorders

Close Encounters of the Oily Kind: Regulation of Transporters by Lipids

Neurotransmitter transporter function can be influenced by a variety of physiological factors, such as temperature, membrane voltage, pH, ion gradients, endogenous ligands, and accessory proteins. Several successful drugs that target transporter proteins, such as the selective reuptake inhibitors, have also been developed. One mechanism for regulating membrane protein function that has only started to make the list has been the impact of lipid environment. Over the last few decades, lipids, the unsung heroes of regulation have begun to emerge as critical modulators of membrane protein function through their direct actions on proteins, through their influence on the lipid milieu and through their roles in signaling pathways

Selenocysteine is transported by EAATs 1–3.

<p><b>A)</b> Representative recordings (upper panel) and averaged normalized transport currents (lower panel) measured at −60 mV as a function of the L-selenocysteine concentration in EAAT3 expressing oocytes (n = 6). Data are presented as the mean and Std. dev. of the mean and fit with the Hill equation to estimate the Km for transport. <b>B)</b> Comparison of the maximal transport currents at −60 mV for L-selenocysteine and L-cysteine by EAAT1 (n>3), EAAT2 (n>5) or EAAT3 (n>10) normalized to the maximal currents induced by L-glutamate measured in the same oocyte. <b>C)</b> Comparison of averaged current-voltage relationships recorded from oocytes expressing EAAT3 for both 1 mM glutamate (red symbols, n = 4) and 1 mM selenocysteine (blue symbols, n = 4). Black symbols indicate the averaged current voltage relationship of the same cells in the absence of substrate (n = 4) and the solid line represents the average of water injected oocytes in the presence of 1 mM glutamate (n = 5).</p

pH affects glutamate inhibition of cysteine transport.

<p>Glutamate inhibition of cysteine uptake from oocytes expressing EAAT3 at three different cysteine concentrations: 30 µM (circles), 300 µM (triangles) and 1 mM (squares) at pH 6.9 (<b>A</b>) and pH 8.5 (<b>B</b>).</p

mEGFPpH detects intracellular pH changes induced by glutamate transport.

<p>Representative image of mEGFPpH transfected HEK293 cells (<b>A</b>) and representative fluorescence traces from HEK293 cells expressing mEGFPpH perfused with 50 mM NH₄Cl (<b>B</b>). Y axis indicates the ratio of fluorescence emission at 510 nm from excitation at 485 nm and 405 nm (F485/F405) (<b>B</b>). Arrows in <b>A</b> indicate the cells from which the traces in <b>B</b> were recorded. <b>C)</b> Fluorescence ratio (F485/F405) as a function of induced intracellular pH following NH₄Cl perfusion (<b>B</b>). <b>D)</b> Perfusion of increasing concentrations of L-glutamate results in increased rate of mEGFPpH fluorescence decrease in HEK293 cells co-transfected with EAAT3 and mEGFPpH. The Y-axis units are the fluorescence ratio for emission at 510 nm with excitation at 485 and 405 nm (F485/F405). <b>E)</b> Perfusion with 100 µM D-aspartate results in intracellular acidification with slope magnitude similar to that for 100 µM L-glutamate (bar graph). Y-axis units are the fluorescence ratio for emission at 510 nm with excitation at 485 and 405 nm (F485/F405). <b>F)</b> Representation of the magnitude of the slope of mEGFPpH fluorescence ratio decrease (left y-axis) as a function of the applied glutamate concentration compared with the glutamate transport activity (right y-axis) in similarly transfected cells.</p

Transport of substrates in cells expressing EAAT2 or EAAT3 differentially affects intracellular pH.

<p>Representative fluorescence recordings from HEK293 cells expressing EAAT2 (<b>A</b>) or EAAT3 (<b>B</b>) in response to short applications of different concentrations of L-cysteine, L-glutamate or L-selenocysteine. The magnitude of maximal steady state slopes (<b>A</b> and <b>B</b>) are plotted in bar graphs below each trace, normalized to the glutamate slope magnitude. <b>C)</b> Representative trace of the effect on cysteine induced mEGFPpH fluorescence changes in EAAT3 expressing HEK293 cells with (left) or without (right) 100 µM TBOA. The Y-axis units for traces represent the fluorescence ratio for emission at 510 nm with excitation at 485 and 405 nm (F485/F405).</p

EAAT3 dependent release of [³H]-L-glutamate or [³⁵S]-L-cysteine.

<p><b>A and B)</b> Release of [³⁵S]-L-Cysteine (<b>A</b>) or [³H]-L-Glutamate (<b>B</b>) from oocytes co-expressing EAAT3 and ASCT, in response to different buffers and conditions <b>C)</b> Averaged current-voltage relationships recorded from oocytes expressing EAAT3 alone (n = 6) or co-expressed with ASCT1 (n = 4) in response to a family of voltage pulses in the absence (black symbols) and the presence of 1 mM serine (blue symbols) or 1 mM glutamate (red symbols). The solid line represents un-injected oocytes in the presence of 1 mM glutamate and 1 mM serine (n = 3).</p

Structures of EAAT substrates and non-substrates.

<p>Diagrams of relevant amino acids and the associated side chain pK_as. Amino acids are depicted in their primary charge state at physiological pH. Abbreviations are: L-Asp, L-aspartate; L-Glu, L-glutamate; L-Sec, L-selenocysteine; L-Cys, L-cysteine; L-Ser, L-serine.</p

Glial and Neuronal Glutamate Transporters Differ in the Na+ Requirements for Activation of the Substrate-Independent Anion Conductance

Excitatory amino acid transporters (EAATs) are secondary active transporters of L-glutamate and L- or D-aspartate. These carriers also mediate a thermodynamically uncoupled anion conductance that is gated by Na+ and substrate binding. The activation of the anion channel by binding of Na+ alone, however, has only been demonstrated for mammalian EAAC1 (EAAT3) and EAAT4. To date, no difference has been observed for the substrate dependence of anion channel gating between the glial, EAAT1 and EAAT2, and the neuronal isoforms EAAT3, EAAT4 and EAAT5. Here we describe a difference in the Na+-dependence of anion channel gating between glial and neuronal isoforms. Chloride flux through transporters without glutamate binding has previously been described as substrate-independent or “leak” channel activity. Choline or N-methyl-D-glucamine replacement of external Na+ ions significantly reduced or abolished substrate-independent EAAT channel activity in EAAT3 and EAAT4 yet has no effect on EAAT1 or EAAT2. The interaction of Na+ with the neuronal carrier isoforms was concentration dependent, consistent with previous data. The presence of substrate and Na+-independent open states in the glial EAAT isoforms is a novel finding in the field of EAAT function. Our results reveal an important divergence in anion channel function between glial and neuronal glutamate transporters and highlight new potential roles for the EAAT-associated anion channel activity based on transporter expression and localization in the central nervous system