Transport Dynamics in a Glutamate Transporter Homologue

Abstract

Glutamate transporters are integral membrane proteins that catalyze neurotransmitter uptake from the synaptic cleft into the cytoplasm of glial cells and neurons 1 . Their mechanism involves transitions between extracellular-(outward-) and intracellular-(inward-) facing conformations, whereby substrate binding sites become accessible to the opposite sides of the membrane 2 . This process has been proposed to entail trans-membrane movements of three discrete transport domains within a trimeric scaffold 3 . Using single-molecule fluorescence resonance energy transfer (smFRET) imaging 4 , we have directly observed large-scale transport domain movements in a bacterial homologue of glutamate transporters for the first time. We find that individual transport domains alternate between periods of quiescence and periods of rapid transitions, reminiscent of bursting patterns first recorded in single ion channels using patch-clamp methods 5,6 . We suggest that the switch to the dynamic mode in glutamate transporters is due to separation of the transport domain from the trimeric scaffold, which precedes domain movements across the bilayer. This spontaneous dislodging of the substrate-loaded transport domain is approximately 100-fold slower than subsequent trans-membrane movements and may be rate determining in the transport cycle. Main TextIn the brain, glutamate mediates excitatory synaptic transmission, responsible for learning, memory formation and cognition 1,7 . Glutamate transporters are electrochemically driven pumps that maintain a low neurotransmitter background at glutamatergic synapses, allowing for repeated rounds of signaling and preventing excitotoxicity 8 . The sodium/aspartate symporter from Pyrococcus horikoshii, Glt Ph , is the only glutamate transporter homologue with known three-dimensional structures of both outward-and inward-facing states 3,9 . Correspondingly, this system has served as a valuable model for establishing the structural and dynamic underpinnings of the transport cycle [10][11][12] . Because Glt Ph originates from a hyper-thermophilic archaeon, it has a slow turnover time of ∼100 seconds at room Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Mechanisms of Anion Conduction by Coupled Glutamate Transporters

“…Whereas the key structural features of secondary active glutamate transport have been established (Akyuz et al, 2013;Crisman et al, 2009;Reyes et al, 2009;Shrivastava et al, 2008), structural and mechanistic details of anion permeation have been hitherto unknown. In this study, we used a combination of computational and experimental approaches to determine how this class of transporters mediates anion permeation through an aqueous conduction pathway.…”

Shared Molecular Mechanisms of Membrane Transporters

“…In Glt Ph , for example, HP2 appears to spontaneously close in the substrate-free state and, therefore, substrate may not serve as a catalyst of the transition. Studies using smFRET, in which the movements of the transport domain have been observed directly, consistently seem to suggest that the translocation of the substrate-loaded transport domain is the slowest step of the transport cycle (137,138). This feature may well be peculiar to Glt Ph , which is a homolog from a hyperthermophilic archaeon.…”

“…A homolog of glutamate transporters from Pyrococcus horikoshii, the sodium-aspartate symporter Glt Ph is a founding member of the group of transporters with an elevator transport mechanism (Figure 2c and Figure 5). Elevator-like movements of the transport domain in Glt Ph have been demonstrated crystallographically (132)(133), by EPR (134,135), and by single-molecule FRET (smFRET) spectroscopy, both in detergent and in liposomes (136)(137)(138). Homotrimeric Glt Ph is composed of a central trimeric scaffold and three peripheral transport domains.…”

Protonation of Glu 135 Facilitates the Outward-to-Inward Structural Transition of Fucose Transporter

“…According to previous experiments on glutamate and aspartate transporters, the large-scale conformational changes of membrane transporters frequently fall in the timescale of seconds (47,48). Such slow molecular events therefore may escape the observing capability of all-atom equilibrium simulations that can only track the timescale of microseconds or perhaps milliseconds at most.…”