Neurotransmitter transporters harness the electrochemical potential gradient of ions, namely sodium, across the cell membrane to import neurotransmitters against their electrochemical gradient into neuronal or glial cells. Effective translocation from extracellular (EC)2
space regulates neuronal signaling by keeping the levels of neurotransmitters sufficiently low at the synapse. Glutamate is the main excitatory neurotransmitter in the central nervous system. Its high concentrations at the synaptic cleft have been linked to neurological diseases such as epilepsy, stroke, ischemia, and Huntington disease (1
). Therefore, glutamate transporters play a key role in preventing excitotoxic effects.
Glutamate transporters belong to the s
arrier 1 (SLC1) family composed of eukaryotic and prokaryotic members that transport acidic or neutral amino acids. The only available high resolution structure for a member of the SLC1 is currently that of the archaeal aspartate transporter from Pyrococcus horikoshii
is a homotrimer. Each monomer is composed of eight transmembrane (TM) helices, TM1–8, and two helical hairpins, HP1 and HP2 (see , A–C
), organized in two structural regions: a transport “core” that binds and transports the substrate and sodium ions and a “scaffold” that provides support for the transport core and forms the intersubunit interface. The scaffold is composed of TM1–6, with the trimerization domain formed by TM2, -4, and -5, and the transport core contains the binding pocket that is composed of TM7, TM8, HP1, and HP2. The hairpins reach from opposite sides of the membrane with their tips coming into very close proximity as has been experimentally shown (see D
FIGURE 1. Structure of the aspartate transporter GltPh.
A displays the trimeric structure in the outward-facing state. B displays one of the subunits with the transport core shown in colored ribbon representation. The transport core is magnified in C and D to highlight (more ...)
has been resolved in several functional states, including outward-facing (OF) (5
) and inward-facing (IF) (7
) states, and an intermediate state (8
). The most prominent difference between the OF and IF states is the almost rigid body translation of the transport core, together with TM3 and -6, by ~15 Å into the cytoplasm, accompanied by rigid body rotation of ~30° in each subunit (see , E
and F). This difference is clearly observed upon structural alignment of the trimerization domains in the OF and IF subunits (7
). We recently proposed that EAAT1 and/or GltPh
would undergo a sequential transition between these two endpoints during the transport cycle, visiting two intermediates composed of 1 (or 2) OF and 2 (or 1) IF subunits (9
). Notably, the recently resolved intermediate structure confirmed this prediction. The latter, composed of two IF subunits and one intermediate OF, with the transport domain shifted by ~3.5 Å and ~15° toward the IF position (8
), overall shows a root mean square deviation of 1.3 Å only from the predicted (9
) 2IF-1OF intermediate.
In all structures resolved in the presence of substrate, the bound aspartate is coordinated by residues on TM7 and -8 and on HP1 and HP2 loops. Additionally, two Na+
-binding sites located ~7 Å from the substrate and from each other have been identified; the first (Na1) is more buried and lies between TM7 and -8, whereas the second (Na2) is located between HP2 and TM7 (5–7) (see , C
and D). These structures are in accord with the topology and function of mammalian and prokaryotic glutamate transporters (10
Despite this significant progress, much remains to be elucidated with regard to the time-resolved events and inter-residue interactions that mediate sodium-coupled substrate binding or release. For instance, the role of HP2 as an EC gate that controls the binding (or unbinding) of substrate and cations in the OF state has been suggested both by the crystallization of the transporter with an antagonist (5
) and by molecular dynamics simulations (16
). This role seems plausible because of its high exposure to the EC environment and ability to move therein unobstructed by the rest of the transporter. On the other hand, the IC gating mechanism is far less clear. Crystallographic (7
) and substrate uptake studies performed by introducing cysteines in a cysteine-less version of EAAT1 (18
) suggested that HP1 might be involved in IC gating, whereas support from time-resolved examinations at an atomic level has been lacking. Here we conducted a series of molecular dynamics runs, using the high performance computing system, Anton, which permits us to examine processes on the microsecond to millisecond timescale (19
) depending on the system size. We were able to view for the first time multiple incidences of IC gate opening and substrate and ion release so as to deduce reproducible patterns and extract statistically reliable information on gating mechanism. The picture that emerged differs from that indirectly inferred from static crystal structures; HP2 (and not HP1) opening is the major event enabling the release of neurotransmitter to the cell interior. HP2 therefore serves as IC gate in the IF state, in addition to its established EC gate role. Our study further highlights the sequence of events that enable the release of substrate, including prior release of Na2 to weaken local interactions and promote substrate dissociation.