Individual Subunits of the Glutamate Transporter EAAC1 Homotrimer Function Independently of Each Other

108

107

Glutamate transporters are essential for recovery of the neurotransmitter glutamate from the synaptic cleft. Crystal structures in the outward-and inward-facing conformations of a glutamate transporter homolog from archaebacterium Pyrococcus horikoshii, sodium/aspartate symporter Glt Ph , suggested the molecular basis of the transporter cycle. However, dynamic studies of the transport mechanism have been sparse and indirect. Here we present highspeed atomic force microscopy (HS-AFM) observations of membranereconstituted Glt Ph at work. HS-AFM movies provide unprecedented real-space and real-time visualization of the transport dynamics. Our results show transport mediated by large amplitude 1.85-nm "elevator" movements of the transport domains consistent with previous crystallographic and spectroscopic studies. Elevator dynamics occur in the absence and presence of sodium ions and aspartate, but stall in sodium alone, providing a direct visualization of the ion and substrate symport mechanism. We show unambiguously that individual protomers within the trimeric transporter function fully independently.Glt Ph | HS-AFM | transporter | elevator mechanism | dynamics

Section: Significancesupporting

confidence: 89%

Direct visualization of glutamate transporter elevator mechanism by high-speed AFM

Ruan

Miyagi

Wang

et al. 2017

108

107

“…This motif has been pointed out to play a role in binding the substrate and sodium ions, as also confirmed by the Glt Ph structure (4,8). In addition, residues in the mammalian EAATs equivalent to the Glt Ph residues D394 and R397 on TM8 and the serine rich motif (S277-S278-S279) at the HP1 tip have been proposed to be involved in glutamate binding, in line with the interactions with the bound aspartate observed in the Glt Ph structure (8)(9)(10).…”

mentioning

confidence: 52%

“…S3). Because the 3 subunits transport the substrate independently (10,31,32) and the core domains undergo uncorrelated dynamics at the timescale of MD simulations (11), we applied pulling forces on the aspartates in each monomer. Simultaneous examination of the 3 aspartates increased the events observed by a factor of 3, thus enhancing the statistical accuracy of our results and permitting us to verify the reproducibility of the results.…”

Section: Steered MD Simulationsmentioning

confidence: 99%

Molecular simulations elucidate the substrate translocation pathway in a glutamate transporter

Shrivastava²,

Amara

et al. 2009

Glutamate transporters are membrane proteins found in neurons and glial cells, which play a critical role in regulating cell signaling by clearing glutamate released from synapses. Although extensive biochemical and structural studies have shed light onto different aspects of glutamate transport, the time-resolved molecular mechanism of substrate (glutamate or aspartate) translocation, that is, the sequence of events occurring at the atomic level after substrate binding and before its release intracellularly remain to be elucidated. We identify an energetically preferred permeation pathway of Ϸ23 Å between the helix HP1b on the hairpin HP1 and the transmembrane helices TM7 and TM8, using the high resolution structure of the transporter from Pyrococcus horikoshii (Glt Ph ) in steered molecular dynamics simulations. Detailed potential of mean force calculations along the putative pathway reveal 2 energy barriers encountered by the substrate (aspartate) before it reaches the exit. The first barrier is surmounted with the assistance of 2 conserved residues (S278 and N401) and a sodium ion (Na2); and the second, by the electrostatic interactions with D405 and another sodium ion (Na1). The observed critical interactions and mediating role of conserved residues in the core domain, the accompanying conformational changes (in both substrate and transporter) that relieve local strains, and the unique coupling of aspartate transport to Na ؉ dislocation provide insights into methods for modulating substrate transport.

“…These transporters thereby maintain an efficient synaptic communication between neurons and prevent extracellular glutamate from reaching neurotoxic levels (1,2). EAATs are trimeric proteins in which each subunit functions as an independent transporter (3,4). Each subunit has eight transmembrane domains and two membrane inserted hair-pin (HP) loops (5)(6)(7).…”

mentioning

confidence: 99%

Evidence for a third sodium-binding site in glutamate transporters suggests an ion/substrate coupling model

Larsson

Wang

Lev

et al. 2010

Excitatory amino acid transporters (EAATs) remove glutamate from synapses. They maintain an efficient synaptic transmission and prevent glutamate from reaching neurotoxic levels. Glutamate transporters couple the uptake of one glutamate to the cotransport of three sodium ions and one proton and the countertransport of one potassium ion. The molecular mechanism for this coupled uptake of glutamate and its co-and counter-transported ions is not known. In a crystal structure of the bacterial glutamate transporter homolog, Glt Ph , only two cations are bound to the transporter, and there is no indication of the location of the third sodium site. In experiments using voltage clamp fluorometry and simulations based on molecular dynamics combined with grand canonical Monte Carlo and free energy simulations performed on different isoforms of Glt Ph as well on a homology model of EAAT3, we sought to locate the third sodium-binding site in EAAT3. Both experiments and computer simulations suggest that T370 and N451 (T314 and N401 in Glt Ph ) form part of the third sodium-binding site. Interestingly, the sodium bound at T370 forms part of the binding site for the amino acid substrate, perhaps explaining both the strict coupling of sodium transport to uptake of glutamate and the ion selectivity of the affinity for the transported amino acid in EAATs.excitatory amino acid transporters | fluorescence | the sodium/aspartate symporter from Pyrococcus horikoshii (Glt Ph ) | simulations G lutamate, the main excitatory neurotransmitter in the central nervous system, is removed from the extracellular synaptic space by the glutamate excitatory amino acid transporters EAAT1-5 (1, 2). These transporters thereby maintain an efficient synaptic communication between neurons and prevent extracellular glutamate from reaching neurotoxic levels (1, 2). EAATs are trimeric proteins in which each subunit functions as an independent transporter (3, 4). Each subunit has eight transmembrane domains and two membrane inserted hair-pin (HP) loops (5-7). EAATs use the Na + and K + gradients in driving the uptake of glutamate against a concentration gradient (8). The uptake of one glutamate is coupled to the cotransport of three Na + ions and one H + ion and the countertransport of one K + ion (9). How the thermodynamically coupled transport of glutamate, H + , Na + , and K + ions is accomplished by glutamate transporters is not known. Here we present evidence for a binding site for Na + ions that suggests a mechanism for the coupling of sodium and glutamate transport.At least one extracellular Na + ion appears to bind before glutamate can bind, and at least one extracellular Na + ion appears to bind after glutamate has bound to the transporter (10-12). For example, in the absence of glutamate, a fluorophore attached to a cysteine at position A430 on HP2 in EAAT3 reported voltageand Na + -dependent fluorescence changes, consistent with Na + binding to the glutamate-free transporter and inducing a conformational change in HP2. Li + also supports glutamat...