The Mechanism of a Neurotransmitter:Sodium Symporter—Inward Release of Na+ and Substrate Is Triggered by Substrate in a Second Binding Site

Shi, Lei; Quick, Matthias; Zhao, Yongfang; Weinstein, Harel; Javitch, Jonathan A.

doi:10.1016/j.molcel.2008.05.008

Cited by 363 publications

(773 citation statements)

References 36 publications

Supporting

Mentioning

696

Contrasting

Unclassified

Order By: Relevance

“…The role of Na2 in sealing the binding site has also been reported in recent simulations of the glutamate transporter (18). Interestingly, a similar role has been proposed for a Na ϩ in a leucine transporter (19).…”

Section: Two Residues Distinguished By Their Persistent Mediating Rolsupporting

confidence: 67%

Molecular simulations elucidate the substrate translocation pathway in a glutamate transporter

Shrivastava²,

Amara

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

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.

show abstract

Section: Two Residues Distinguished By Their Persistent Mediating Rolsupporting

confidence: 67%

Molecular simulations elucidate the substrate translocation pathway in a glutamate transporter

Shrivastava²,

Amara

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…Taken together, our findings provide more evidence against the supposition that there is a second or S2 high-affinity substrate binding site in LeuT [18][19][20][21][22]. The results of the Javitch group related to the S2 site, the properties of the F253A mutant and the role of the putative S2 site in the mechanism of LeuT and NSSs are, at present, without satisfactory explanation.…”

Section: Discussionmentioning

confidence: 49%

Substrate binds in the S1 site of the F253A mutant of LeuT, a neurotransmitter sodium symporter homologue

Wang

Gouaux

2012

EMBO Reports

View full text Add to dashboard Cite

LeuT serves as the model protein for understanding the relationships between structure, mechanism and pharmacology in neurotransmitter sodium symporters (NSSs). At the present time, however, there is a vigorous debate over whether there is a single high-affinity substrate site (S1) located at the original, crystallographically determined substrate site or whether there are two high-affinity substrates sites, one at the primary or S1 site and the other at a second site (S2) located at the base of the extracellular vestibule. In an effort to address the controversy over the number of high-affinity substrate sites in LeuT, one group studied the F253A mutant of LeuT and asserted that in this mutant substrate binds exclusively to the S2 site and that 1 mM clomipramine entirely ablates substrate binding to the S2 site. Here we study the binding of substrate to the F253A mutant of LeuT using ligand binding and X-ray crystallographic methods. Both experimental methods unambiguously show that substrate binds to the S1 site of the F253A mutant and that binding is retained in the presence of 1 mM clomipramine. These studies, in combination with previous work, are consistent with a mechanism for LeuT that involves a single high-affinity substrate binding site.

show abstract

“…2) (10). A sodium ion was assigned to this density and remained bound in 100-ns MD simulations of this outward-occluded state (11), similar to the behavior of Na2 in simulations of LeuT (25)(26)(27). In a structure of the inward-facing state, in contrast, helix B3 is ∼4 Å further away from A1, so that the sodium-binding site appears to be no longer intact (11); indeed, in multiple MD simulations of the inward-facing state, sodium did not remain bound to this site longer than 2 ns (11), as is consistent with the observations discussed above for LeuT.…”

mentioning

confidence: 65%

“…Binding of 22 Na + to purified BetP variants was performed by means of the SPA as described (26). Experiments were preformed in 0.05-1.05 M Tris/ MES (pH 7.5), 1.05-0.05 M NaCl (equimolar replacement of Tris/MES with NaCl), 20% (vol/vol) glycerol, 1 mM Tris(2-carboxyethyl)phosphine, 0.1% ndodecyl-β-D-maltopyranoside using 100 ng of purified BetP variants, 2.5 mg/ mL streptavidin-coated YSi SPA beads (RPNQ0012; Perkin Elmer), and 1 μM of [ 22 Na]Cl (19.1 Ci/mmol) (Perkin Elmer).…”

Section: Methodsmentioning

confidence: 99%

Investigation of the sodium-binding sites in the sodium-coupled betaine transporter BetP

Khafizov¹,

Pérez²,

Koshy³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

Sodium-coupled substrate transport plays a central role in many biological processes. However, despite knowledge of the structures of several sodium-coupled transporters, the location of the sodiumbinding site(s) often remains unclear. Several of these structures have the five transmembrane-helix inverted-topology repeat, LeuTlike (FIRL) fold, whose pseudosymmetry has been proposed to facilitate the alternating-access mechanism required for transport. Here, we provide biophysical, biochemical, and computational evidence for the location of the two cation-binding sites in the sodium-coupled betaine symporter BetP. A recent X-ray structure of BetP in a sodium-bound closed state revealed that one of these sites, equivalent to the Na2 site in related transporters, is located between transmembrane helices 1 and 8 of the FIRL-fold; here, we confirm the location of this site by other means. Based on the pseudosymmetry of this fold, we hypothesized that the second site is located between the equivalent helices 6 and 3. Molecular dynamics simulations of the closed-state structure suggest this second sodium site involves two threonine sidechains and a backbone carbonyl from helix 3, a phenylalanine from helix 6, and a water molecule. Mutating the residues proposed to form the two binding sites increased the apparent K m and K d for sodium, as measured by betaine uptake, tryptophan fluorescence, and 22 Na + binding, and also diminished the transient currents measured in proteoliposomes using solid supported membrane-based electrophysiology. Taken together, these results provide strong evidence for the identity of the residues forming the sodium-binding sites in BetP.secondary transport | symmetry | membrane protein | alkali metal ion | osmoregulation

show abstract

The Mechanism of a Neurotransmitter:Sodium Symporter—Inward Release of Na+ and Substrate Is Triggered by Substrate in a Second Binding Site

Cited by 363 publications

References 36 publications

Molecular simulations elucidate the substrate translocation pathway in a glutamate transporter

Molecular simulations elucidate the substrate translocation pathway in a glutamate transporter

Substrate binds in the S1 site of the F253A mutant of LeuT, a neurotransmitter sodium symporter homologue

Investigation of the sodium-binding sites in the sodium-coupled betaine transporter BetP

Contact Info

Product

Resources

About