Biomedical Engineering Reference
In-Depth Information
synapse of their release, thereby minimizing chemical crosstalk between adjacent synapses. Third,
transporters allow recycling by reuptake of transmitters into the nerve terminal with presumed sav-
ings in synthetic cost. The crucial physiological role of the neurotransmitter transporters has been
cemented by gene knockout experiments. In case of, e.g., the dopamine transporter (DAT), the dis-
ruption of the transporter gene in mice revealed the unequivocal importance of this carrier in the control
of locomotion, growth, lactation, and spatial cognitive function.
The SLC6 transporters include not only the transporters of neurotransmitters (Table 14.1,
Figure 14.1) but also transporters of amino acids, metabolites (creatine), and osmolytes (betaine and
taurine) (Figure 14.1). Moreover, a large number of homologues have been identii ed in archaea and
bacteria. The function of the majority of these transporters is still unknown; however, a few of them
have been identii ed as amino acid transporters, such as, e.g., the leucine transporter LeuT Aa from
the Aquifex aeolicus bacterium and the tyrosine transporter Tyt1 from Fusobacterium nucleatum .
At the molecular level, the SLC6 family transporters operate as Na + dependent cotransporters that
utilize the transmembrane Na + gradient to couple the “downhill” transport of Na + with the “uphill”
transport (against a concentration gradient) of their substrate from the extracellular to the intracel-
lular environment. The transport process is so efi cient that, e.g., the serotonin transporter (SERT)
can accumulate internal serotonin (5-HT) to concentrations 100-fold higher than the external
medium when appropriate ion gradients are imposed. Most SLC6 transporters are also cotransport-
ers of Cl and, accordingly, SLC6 transporters have been referred to as the family of Na + /Cl -dependent
transporters.
14.2.1 S TRUCTURES AND M ECHANISMS OF SLC6 T RANSPORTERS
It is generally believed that SLC6 transporters function according to an alternating access model,
which suggests a transport mechanism in which, at any given time, only the substrate-binding site is
accessible to either the intracellular or the extracellular side of the membrane. Thus, at all times an
impermeable barrier exists between the binding site and one side of the membrane, but the barrier
can change from one side of the binding site to the other, giving the site alternate access to the two
aqueous compartments that the membrane separates. A prerequisite for this model is the existence
of both external and internal “gates,” i.e., protein domains that are capable of occluding access to the
binding site of the substrate from the external and internal domains, respectively (Figure 14.2).
In the absence of high-resolution structural information, however, it was, for a long time, only
possible to speculate about the molecular basis of the transport process. A major breakthrough came
when the bacterial homologue, LeuT Aa , which displays 20%-25% sequence identity to its mamma-
lian counterparts, was successfully crystallized and the structure solved at high resolution (1.65 Å).
The transporter was crystallized with substrate and Na + bound to the transporter. The x-ray diffrac-
tion pattern revealed a protein containing 12 transmembrane segments (TMs) in a unique fold and
with a binding site for l-leucine buried inside the center of the protein (Figure 14.3). The diffraction
pattern also revealed an unexpected structural repeat in the i rst 10 TMs that relates TM1-5 with
TM6-10 around a pseudo-twofold axis of symmetry located in the plane of the membrane. The
binding pockets for leucine and Na + are formed by TM1, TM3, TM6, and TM8. TM3 and TM8 are
long helices that are related by the twofold symmetry axis and are strongly tilted (~50°) (Figure
14.3). TM1 and TM6 are characterized by unwound breaks in the helical structure in the middle of
the lipid bilayer. These breaks expose main carbonyl oxygen and nitrogen atoms for direct interaction
with the substrate.
The LeuT Aa structure was crystallized in a conformation in which access to the substrate-binding
site is closed from both the intracellular and extracellular environments, i.e., the predicted external
and internal “gates” appear closed in the structure, and, hence, the structure likely represents an
intermediate state between the outward facing conformation (where the substrate-binding site is
exposed to the extracellular environment) and the inward facing conformation (where it is exposed
to the intracellular milieu).
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