Biomedical Engineering Reference
In-Depth Information
the leucine-binding site is that it exposes main-chain atoms from the unwound regions of TM1 and
TM6 make most of the contacts with the
-carboxy groups of the bound leucine. The
unwound regions of TM1 and TM6 allow direct hydrogen-bonding partners as well as orientating
the
α
-amino and
α
α
-amino and
α
-carboxy groups so they can bind close to the ends of the helical segments and
-helix dipole interactions. In addition and as mentioned earlier, one of the Na + ions makes
direct contact with the carboxyl group of the leucine. Notably, this interaction cannot occur in
transporters of the biogenic amines, dopamine, 5-HT, and norepinephrine, which do not possess a
carboxyl group; however, the glycine in position 24 of LeuT Aa (which is conserved among the amino
acid transporters) is replaced with aspartate in these transporters (Asp79 in the DAT). According to
the LeuT Aa structure, this aspartate is predicted to be in the immediate proximity of Na + and, thus,
can probably substitute for the missing carboxyl group of the substrate. Studies on DAT and SERT
also support the hypothesis that the aspartate in TM1 plays an important role in the coordination
of the protonated primary amine in dopamine, and 5-HT. In GAT-1 the residue in this position is a
glycine as it is in the LeuT Aa . Hence, GABA is probably coordinated in the same way as leucine in
the LeuT Aa . Indeed, the residue seems to be important for the binding of GABA to the GAT-1.
Right above the leucine molecule in the LeuT Aa structure, a tyrosine in TM3 (Tyr108) forms via
its hydroxyl a hydrogen bond with the main-chain amide nitrogen of Leu25 in TM1. This interaction
could function as a latch to stabilize the irregular structure near the unwound region in TM1 and
may even be the i rst determinant of the closure of the extracellular gate. This hypothesis is even
more interesting in light of the fact that the tyrosine is strictly conserved among all SLC6 family
members and has been implicated in the substrate binding and transport of GAT-1 and SERT. Of
further interest, recent homology modeling studies in DAT and SERT suggest that in these trans-
porters, the hydroxyl group of the tyrosine does not form a hydrogen bond with the main-chain
amide nitrogen of Leu25 but with the central aspartate in TM1, believed also to interact directly
with the monoamine substrates (Asp79 in DAT and Asp98 in SERT and corresponding to position
24 in LeuT Aa ). In support of this hypothesis and thereby of the role of the hydrogen bond in stabiliz-
ing the substrate-binding site, mutation of the tyrosine in DAT (Tyr156) to phenylalanine decreases
apparent dopamine around 10-fold and decreases the maximum uptake capacity by ~50%.
It is also important to note that the identii cation of residues shaping the leucine-binding site in
LeuT Aa illuminates the determinants of substrate specii city in the eukaryotic homologues. In the
SERT, for example, residues at equivalent positions to those surrounding the isopropyl moiety of
leucine in LeuT Aa are replaced with smaller amino acids to accommodate the larger serotonin mol-
ecule. Correspondingly, homology modeling of the glycine transporters GluT1 and GlyT2 suggests
together with mutational analysis that the substrate specii city is determined by a few key residues
and that the ability of GlyT1 but not GlyT2 to transport sarcosine in addition to glycine is deter-
mined by a single residue difference between GlyT1 and GlyT2.
establish
α
14.3 DRUGS TARGETING BIOGENIC AMINE TRANSPORTERS:
SPECIFICITY, USE, AND MOLECULAR MECHANISMS OF ACTION
The biogenic amine transporters, DAT, NET, and SERT, are targets for a wide variety of drugs.
Overall, these drugs can be classii ed as either pure inhibitors that block substrate binding and
transport, or as substrates that in addition to competing with the endogenous substrate are also
transported themselves.
14.3.1 C OCAINE , B ENZTROPINE , AND O THER T ROPANE C LASS I NHIBITORS
The most thoroughly studied class of inhibitors of biogenic amine transporters is the “tropane”
class, with cocaine as the most well-known member (Figure 14.4). Cocaine is a moderately potent
antagonist inhibiting the function of all three transporters nonselectively. However, earlier correlative
studies as well as studies on genetically modii ed mice suggest that presynaptic DAT is the primary
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