Molecular Mechanism of Ion-Ion and Ion-Substrate Coupling
in the Na1-Dependent Leucine Transporter LeuT
David A. Caplan,*y Julia O. Subbotina,*y and Sergei Yu Noskov*y
*Institute for Biocomplexity and Informatics, and yDepartment of Biological Sciences, University of Calgary, Calgary, Alberta, Canada
ABSTRACT Ion-coupled transport of neurotransmitter molecules by neurotransmitter:sodium symporters (NSS) play an
important role in the regulation of neuronal signaling. One of the major events in the transport cycle is ion-substrate coupling
and formation of the high-affinity occluded state with bound ions and substrate. Molecular mechanisms of ion-substrate coupling
and the corresponding ion-substrate stoichiometry in NSS transporters has yet to be understood. The recent determination of a
high-resolution structure for a bacterial homolog of Na1/Cl-dependent neurotransmitter transporters, LeuT, offers a unique
opportunity to analyze the functional roles of the multi-ion binding sites within the binding pocket. The binding pocket of LeuT
contains two metal binding sites. The first ion in site NA1 is directly coupled to the bound substrate (Leu) with the second ion in
the neighboring site (NA2) only ;7 A˚ away. Extensive, fully atomistic, molecular dynamics, and free energy simulations of LeuT
in an explicit lipid bilayer are performed to evaluate substrate-binding affinity as a function of the ion load (single versus double
occupancy) and occupancy by specific monovalent cations. It was shown that double ion occupancy of the binding pocket is
required to ensure substrate coupling to Na1 and not to Li1 or K1 cations. Furthermore, it was found that presence of the ion in
site NA2 is required for structural stability of the binding pocket as well as amplified selectivity for Na1 in the case of double ion
occupancy.
INTRODUCTION
Neurotransmitter transporters regulate the concentrations of
specific neurotransmitters within the synaptic cleft (1,2).
They are typically located in pre/post-synaptic and glial cell
membranes and are responsible for rapidly clearing the
neurotransmitters from the synapse. Many neurotransmitter
transporters rely upon the electrochemical gradient of ions
across the membrane (plasma or vesicular) to drive the uphill
transport of neurotransmitters across membranes. A large set
of transporters can be classified as neurotransmitter:sodium
symporters (NSS), which utilize the sodium cation electro-
chemical gradient to drive transport. The NSS family repre-
sents some of the most well-studied transporters and contains
proteins specific to dopamine, 5-HT, norepinephrine, gly-
cine, and GABA. Transporters in this family have been as-
sociated with a large number of disorders including
depression, schizophrenia, irritable bowel syndrome, and
Parkinson’s disease (3–5). These transporters (most notably
the human serotonin transporter, hSERT) are also extremely
common drug targets (3). For example, hSERT is targeted by
a panel of antidepressant drugs as well as by narcotics such as
MDMA (‘‘ecstasy’’) and cocaine, in addition to stimulants
such as amphetamines. Much of the current knowledge about
transporters comes from analysis of genetic data and from
pharmacological studies (dose-response, binding, and trans-
port assays).
In 2005, Yamashita et al. published the first crystal struc-
ture (PDB ID 2A65) of a bacterial homolog of Na1-Cl-
dependent neurotransmitter transporters (6) and opened a
new avenue for discovering transporter structure/function
relationships. The leucine transporter (LeuT) comes from the
prokaryotic organism Aquifex aeolicus, which lives around
deep sea vents where the temperatures average 97C. The
overall sequence identity between LeuT and related eukary-
otic transporters (such as hSERT, NET, and DAT) ranges
between 20 and 25%. Despite the low overall sequence
identity, it has been shown that several functional regions
(such as the active site) are highly conserved throughout the
family (6,7). More recently, the same group published crystal
structures (PDB IDs 2Q6H, 2Q72, 2QB4, and 2QEI) of LeuT
in complex with three antidepressant drugs (clomipramine,
imipramine, and desipramine) (6).
The active site of LeuT contains a substrate (leucine or
alanine) binding site as well as two sodium-binding sites
labeled NA1 and NA2 (8). One interesting aspect of the ac-
tive site is how one of the sodium ions (NA1) is coordinated
by the leucine substrate in addition to a transmembrane
component (TM6). The other sodium ion (NA2) is coordi-
nated by five residues that form part of a helix-break-helix
motif of TM1. It is thought that the sodium ions are required
to organize the substrate-binding site partially formed by
flexible transmembrane helices (TM1 and TM6) (8). The
crystal structures of LeuT show two bound sodium ions,
suggesting that the binding stoichiometry is 2.We have yet to
determine transport stoichiometry since it is difficult to ac-
curately measure experimentally. It is therefore unknown
whether both ions are required for both binding and transport.
Some related transporters (GABA and glycine transporters)
are known to transport two sodium ions with each substrate
and others (SERT) are known to transport one sodium ion
doi: 10.1529/biophysj.108.139741
Submitted June 10, 2008, and accepted for publication August 5, 2008.
Address reprint requests to Sergei Noskov, Tel.: 403-210-7971; E-mail:
snoskov@ucalgary.ca.
Editor: Peter Tieleman.
 2008 by the Biophysical Society
0006-3495/08/11/4613/09 $2.00
Biophysical Journal Volume 95 November 2008 4613–4621 4613