Biophysical Journal Volume 106 June 2014 2547–2548 2547

New and Notable

Coupling betweenNeurotransmitter Translocationand Protonation State of aTitratable Residue duringNaD-Coupled Transport

Ivet Bahar*Department of Computational and SystemsBiology, School of Medicine, University ofPittsburgh, Pittsburgh, Pennsylvania

The functional significance of excit-atory amino-acid transporters (EAATs)in clearing excess glutamates fromcentral nervous system synapses iswidely recognized. An archaeal aspar-tate transporter, GltPh, fromPyrococcushorikoshii has broadly served as amodel system for gaining structure-based insights into the mechanism offunction of human EAATs, as the firststructurally resolved member (1) ofthe family of glutamate transporters.GltPh has since been resolved in multi-ple states, which helped us gain insightsinto the structural basis of the interac-tions involved in transport activity.Yet, translation of structural and dy-namic data obtained by structural andcomputational studies for the archaealtransporter to human EAATs (subtypeshEAAT1-5) has been a challenge,partly due to the moderate sequencesimilarity (30–40%) between theseorthologs, and the differences intheir functional mechanisms. Recently,another ortholog, aspartate transporter,GltTk from Thermococcus kodakaren-sis has been resolved in substrate-freeform, providing new insights into themechanism of reconfiguration back toan outward-facing state after the releaseof substrate and Naþ ions (2).

Glutamate transport is a typicalexample of secondary transport pro-cess, fueled by the cotransport of so-dium ions down their electrochemical

http://dx.doi.org/10.1016/j.bpj.2014.05.011

Submitted April 30, 2014, and accepted for

publication May 5, 2014.

*Correspondence: bahar@pitt.edu

Editor: Bert de Groot.

� 2014 by the Biophysical Society

0006-3495/14/06/2547/2 $2.00

gradient. EAATs (hEAAT1-3) alsocotransport a proton, in addition tothree Naþ ions. Structure-based com-putations helped in recent years inelucidating the membrane-mediatedmechanisms that permit the alternatingaccess of the transporter between out-ward- and inward-facing states (3),the sequence of events during ion-coupled binding of substrate (4), andthe position of sodium-binding sites(5,6). Yet, the mechanism of couplingbetween substrate release and protoncotransport is yet to be established.EAATs further function as chloridechannels, and the transport cycle iscompleted by the countertransportof a Kþ ion, providing additionallayers of complexity in neurotrans-mitter transport process.

Coupling between the protonationstate of a titratable residue near sub-strate/ion-binding site and substratebinding/release has been recentlynoted in a number of sodium/proton-coupled transporters (7–9). The pro-tonation of an acidic ion may (partly)shield the unfavorable electrostaticinteractions with a negatively chargedsubstrate near bound substrate, andsubstrate release may be succeededby the deprotonation of the titratableresidue upon exposure to aqueousenvironment. Similar phenomena havebeen observed by Heinzelmann andKuyucak (10). Using homologymodels created for the outward-and inward-facing conformations ofEAAT3, they confirmed with the helpof free energy perturbation and ther-modynamic integration methods thata glutamate, E373, is the most prob-able candidate for carrying the cotrans-ported proton. The study further shedslight to a sequence of events and poten-tial involvement of Kþ in enablingproton cotransport and resuming thetransport cycle. E373 is protonated inthe presence of substrate and one ofthe sodium ions (Na2). Its protonationfavors neurotransmitter uptake andbinding in the outward-facing state.In the inward-facing state, on the otherhand, the dislocation of neurotrans-

mitter and Na2, and the opening ofthe HP2 gate, prompt the release ofneurotransmitter and the exposureof E373 to intracellular water, thusreducing the pKa near the bindingpocket in favor of the deprotonationof E373. Proton release is completedupon binding of a Kþ ion to the samesite. Binding of Kþ to the same site isconsistent with newly resolved GltTkstructure, which showed that the vicin-ity of GltTk Q321 (counterpart of E373in EAAT3) could form a Kþ bindingsite (2).

The possible involvement of EAAT3E373 as a proton acceptor has beenproposed almost a decade ago (11),and the equivalent residue (E404) inthe rat homolog of EAAT2 has beenproposed to bind Kþ in 1997 (12).These observations confirm these orig-inal studies, consistent with the conser-vation of glutamate at this particularsequence position across hEAAT sub-types. The originality of the studylies, however, in the quantitativedemonstration of the following:

1. The high affinity of E373 for beingprotonated in the outward-facingstate,

2. Its effect on stabilizing the boundsubstrate,

3. The coupling of its protonation stateto neurotransmitter release, and

4. The effect of bound cations (Naþ orKþ) in modulating its protonationstate.

The pKa values reported in Table 2 inHeinzelmann and Kuyucak (10) for aseries of titratable residues near thebinding pocket highlight the distinctiveprotonation propensities of E374.

Notably, in GltPh as well as GltTk,the same position is occupied by aglutamine, Q318 and Q321, respec-tively. The archaeal transporter doesnot require cotransporting of a proton,nor the countertransport of a Kþ ion(13,14). Notably, the terminal -OHgroup of glutamate (protonated E373)

2548 Bahar

in hEAATs is replaced by an NH2

group (Q318) in GltPh. The involve-ment of Q318 in interactions that con-trol the gating motions of the HP2 loophas been noted earlier (15), and micro-seconds simulations revealed the keyrole of HP2 in both extracellular andintracellular gating (16), in supportof the involvement of this particularsite in mediating substrate uptake andrelease.

The integrated molecular dynamics-free energy perturbation/thermody-namic integration approach adoptedby Heinzelmann and Kuyucak (10)provides a comprehensive frameworkfor assessing the protonation/deproto-nation propensities of titratable resi-dues that may (directly or indirectly)affect substrate-binding or -release.Compared to continuum calculationsof pKa, the method presents the advan-tage of providing a full-atomic descrip-tion of the coupled dynamics of thetransporter and environment, and maybe applicable to proton-coupled trans-location events in general.

REFERENCES

1. Yernool, D., O. Boudker, ., E. Gouaux.2004. Structure of a glutamate transporter

Biophysical Journal 106(12) 2547–2548

homologue from Pyrococcus horikoshii. Na-ture. 431:811–818.

2. Jensen, S., A. Guskov, ., D. J. Slotboom.2013. Crystal structure of a substrate-freeaspartate transporter. Nat. Struct. Mol. Biol.20:1224–1226.

3. Lezon, T. R., and I. Bahar. 2012. Constraintsimposed by the membrane selectively guidethe alternating access dynamics of the gluta-mate transporter GltPh. Biophys. J. 102:1331–1340.

4. Huang, Z., and E. Tajkhorshid. 2008. Dy-namics of the extracellular gate and ion-sub-strate coupling in the glutamate transporter.Biophys. J. 95:2292–2300.

5. Larsson, H. P., X. Wang, ., S. Y. Noskov.2010. Evidence for a third sodium-bindingsite in glutamate transporters suggests anion/substrate coupling model. Proc. Natl.Acad. Sci. USA. 107:13912–13917.

6. Huang, Z., and E. Tajkhorshid. 2010. Identi-fication of the third Naþ site and thesequence of extracellular binding eventsin the glutamate transporter. Biophys. J.99:1416–1425.

7. Zomot, E., and I. Bahar. 2011. Protonationof glutamate 208 induces the release of ag-matine in an outward-facing conformationof an arginine/agmatine antiporter. J. Biol.Chem. 286:19693–19701.

8. Mwaura, J., Z. Tao, ., C. Grewer. 2012.Protonation state of a conserved acidicamino acid involved in Naþ binding to theglutamate transporter EAAC1. ACS Chem.Neurosci. 3:1073–1083.

9. Cheng, M. H., and R. D. Coalson. 2012.Molecular dynamics investigation of Cl�

and water transport through a eukaryotic

CLC transporter. Biophys. J. 102:1363–1371.

10. Heinzelmann, G., and S. Kuyucak. 2014.Molecular dynamics simulations elucidatethe mechanism of proton transport in theglutamate transporter EAAT3. Biophys. J.106:2675–2683.

11. Grewer, C., N. Watzke, ., A. Bicho. 2003.Is the glutamate residue Glu-373 the protonacceptor of the excitatory amino acid carrier1? J. Biol. Chem. 278:2585–2592.

12. Kavanaugh, M. P., A. Bendahan, ., B. I.Kanner. 1997. Mutation of an amino acidresidue influencing potassium coupling inthe glutamate transporter GLT-1 inducesobligate exchange. J. Biol. Chem. 272:1703–1708.

13. Ryan, R. M., E. L. Compton, and J. A. Mind-ell. 2009. Functional characterization of aNaþ-dependent aspartate transporter fromPyrococcus horikoshii. J. Biol. Chem. 284:17540–17548.

14. Groeneveld, M., and D. J. Slotboom. 2010.Naþ:aspartate coupling stoichiometry inthe glutamate transporter homologue GltPh.Biochemistry. 49:3511–3513.

15. Shrivastava, I. H., J. Jiang, ., I. Bahar.2008. Time-resolved mechanism of extra-cellular gate opening and substrate bindingin a glutamate transporter. J. Biol. Chem.283:28680–28690.

16. Zomot, E., and I. Bahar. 2013. Intracellulargating in an inward-facing state of aspartatetransporter GltPh is regulated by the move-ments of the helical hairpin HP2. J. Biol.Chem. 288:8231–8237.