br Recent insights into EAAT structure function

Recent insights into EAAT structure/function aspects The EAATs are secondary-active transporters coupling the movement of one Glu with the symport of three Na+ and one proton and the counter-transport of one K+ [3, 4]. The transporters are trimeric assemblies of protomers that function independently [5, 6], and although most EAATs are assembled as homotrimers, the recent in vitro demonstration of heterotrimeric EAAT3/EAAT4 formation indicate that native EAAT populations may comprise more than five trimeric assemblies [7]. Crystal structures of the prokaryotic EAAT homologues GltPh or GltTk, sodium/aspartate symporters from Pyrococcus horikoshii and Thermococcus kodakarensis, have revealed that each protomer consists of an immobile trimerization domain and a transport domain that encompasses the substrate, Na+, H+ and K+ PSB 1115 (Figure 1a). The trimerization domain mediates inter-protomeric interactions in the trimer and constitutes a rigid scaffold for the dramatic movements of the transport domain during the transport process (Figure 1b,c). The transport process. During the last decade high resolution structures of GltPh and GltTk in multiple conformations have provided highly detailed information about the structural transformations during the transport cycle (outlined in Figure 1c, reviewed in [3, 8]). Importantly, recent fluorescence spectroscopy studies have provided insight into the dynamics underlying these conformational changes, thus breathing life into the structures. Perhaps the most interesting observations to come out of these studies are the insights into the driving force of the transport process and the anarchistic nature of operations in the trimer. As outlined in Figure 1c, substrate binding to the transporter is coupled to the binding of all three Na+ in a highly cooperative manner at both the outward-facing and inward-facing conformations []. Binding of the initial two Na+ as well as of the third Na+ to GltPh have been shown to be associated with great energy costs, indicative of the ions inducing major rearrangements in the substrate binding site rather than binding to pre-existing sites []. Thus, the key role of Na+ in the transport process appears to be routed in its induction of a binding site capable of binding the substrate and of undergoing the transmembrane movement of the transport domain the rather than stabilizing any of these subsequent structural transitions. Analogously, K+ binding to the inward-facing EAAT conformation has been proposed to enable the re-translocation of the substrate-free transport domain to the outward-facing conformation [10]. Two recent single-molecule FRET studies of GltPh have reported transport events mediated by the three protomers in the trimer to take place in an uncoordinated and stochastic manner [11••, 12••]. Thus, while trimeric EAAT assembly undoubtedly is essential for cell membrane expression and stability, the transport seems to be orchestrated by completely independent units tightly regulated by cation binding and unbinding events. EAATs as anion channels. EAATs are not only secondary-active Glu transporters, but also anion-selective channels and thus represent prototypical dual function membrane transport proteins [13, 14]. EAAT anion conduction is mediated by a perfectly anion-selective conduction pathway and exhibits unitary current amplitudes similar to those of specialized anion channels [15]. Macroscopic EAAT anion currents are substrate-dependent and voltage-dependent and follow closely transitions within the uptake cycle [16]. Noise analysis has revealed unitary current amplitudes that are identical in the presence as well as in the absence of Glu [17]. These results support the notion that anion permeation occurs through a defined ion conduction pathway that is opened and closed by conformational changes coupled to the transport process rather than by multiple distinct conduction pathways associated with different transporter states.