The Chloride Permeation Pathway of a Glutamate Transporter and Its Proximity to the Glutamate Translocation Pathway

Abstract

Excitatory amino acid transporters (EAATs) regulate glutamate concentrations in the brain to maintain normal excitatory synaptic transmission. A widely accepted view of transporters is that they consist of a pore with alternating access to the intracellular and extracellular solutions, which serves to couple ion movement to the movement of substrate. However, recent observations that EAATs, and also a number of other neurotransmitter transporters, can also function as ligand-gated chloride channels have blurred the distinctions between transporters and ion channels. Here we show that mutations in the second transmembrane domain (TM2) of EAAT1 alter anion permeation properties without affecting glutamate transport and that a number of TM2 residues are accessible to the external aqueous solution. Furthermore, we demonstrate that the extracellular edge of TM2 is in close proximity to a membrane-associated domain that influences glutamate transport. This study will provide the foundation for beginning to understand how transporters can function as both transporters and ion channels.In the mammalian central nervous system, glutamate is the predominant excitatory neurotransmitter, and EAATs 1 regulate synaptic glutamate concentrations to maintain a dynamic signaling process between neurons. EAATs are secondary active transporters in which glutamate is co-transported with 3 Na ϩ and 1 H ϩ followed by the counter-transport of 1 K ϩ (1), which enables EAATs to maintain a 10 6 -fold glutamate concentration gradient across the cell membrane. In addition to this coupled transport conductance, glutamate also activates a thermodynamically uncoupled chloride conductance through the transporter (2-4), which may serve a number of functions such as modulation of cell excitability, regulation of the rate of voltage-dependent glutamate uptake, and influencing ion homeostasis (4 -7). A number of other neurotransmitter transporters, including transporters for dopamine, serotonin, noradrenaline, and ␥-aminobutyric acid, also allow uncoupled fluxes of ions (8 -11), and in the case of the dopamine transporter the substrate-activated chloride conductance regulates presynaptic excitability (8). At present there is very little understanding of the molecular basis for how transporters can support these separate functions.Five EAAT subtypes have been characterized (3,(12)(13)(14)(15)(16), and the highly conserved carboxyl-terminal half of EAATs has been identified, by the use of chimeric transporters and site-directed mutagenesis, as forming the glutamate binding and/or translocation domain (17-23) (see Fig. 1A). However, a number of mutations that disrupt transport do not affect chloride permeation (for example see Refs. 18 and 24), and several recent studies have reported selective modulation of the transport component without affecting the chloride component of transport (20 -22), implying there are separate molecular determinants for glutamate transport and chloride permeation. In order to investigate this idea further, it is necessary t...

Structure of a glutamate transporter homologue from Pyrococcus horikoshii

“…Residues in eukaryotic transporters that form disulphide bonds when mutated to cysteines are boxed and the bonds are indicated by dashed lines 13,29 . Insertions in eukaryotic transporters between helices 4b and 4c are not included and are marked by XXX; the longer N and C termini of eukaryotic transporters are also not included.…”

“…1). Residues in the carboxy-terminal half of eukaryotic and prokaryotic transporters are crucial for substrate binding, substrate transport and ion coupling (for recent reviews, see refs [10][11][12], whereas residues in the amino-terminal portion of the eukaryotic transporters are implicated in the thermodynamically uncoupled chloride flux 13 . Determination of the transmembrane topology of glutamate transporters has been fraught with uncertainty, and there are multiple models, each possessing non-canonical elements of transmembrane protein structure [14][15][16][17][18][19][20] .…”

The leak mode of type II Na+-Pi cotransporters

“…These studies establish a direct link at the molecular level between the leak current and the heterologously expressed protein (reviewed in refs. [26][27][28][29][30][31][32][33][34]. The most convincing approach to confirm the intrinsic nature of the leak current is to purify and reconstitute the protein in a membrane preparation.…”

Disulfide Cross-linking of a Multidrug and Toxic Compound Extrusion Transporter Impacts Multidrug Efflux

“…Cysteine Cross-linking and Gel Mobility Shift Assay-Purified NorM-NG variants at 10 M were incubated with 50 or 200 M CuSO 4 and 100 or 400 M 1,10-phenanthroline in 20 mM HEPES, pH 7.5, 0 or 100 mM NaCl, 0 or 1 mM tetraphenylphosphonium (TPP), 20% glycerol, and 0.05% n-dodecyl-␤-maltoside buffer for 10 min at 22°C (12)(13)(14)(15)(16)(17). 20 mM N-(5-fluoresceinyl)maleimide was then added to the samples and left for 10 min at 22°C in the dark.…”