Biomedical Engineering Reference
In-Depth Information
Taken together, based on the LeuT
Aa
structure and available functional data, it seems reasonable
to conclude that SLC6 transporters follow an alternating access model. However, the mechanism
of transport by LeuT
Aa
is probably distinct from those suggested for other ion-coupled transporters;
hence, the mechanistic predictions clearly differ from those involving movements of two sym-
metrical hairpins reaching from the extracellular and intracellular environments, respectively, that
were offered for sodium-coupled glutamate transporters based on a crystal structure of a bacterial
member of this transporter family. Similarly, the suggested mechanism differs from the “rocker-
switch” type mechanism proposed for Lac permease and the glycerol-3-phosphate transporter,
two other recently crystallized transport proteins that mediate proton-coupled secondary active
transport.
14.2.2 T
HE
B
INDING
S
ITES
FOR
N
A
+
AND
C
L
−
: I
MPORTANCE
IN
S
UBSTRATE
B
INDING
AND
T
RANSLOCATION
The binding and cotransport of sodium ions is a feature that probably serves several purposes: i rst,
the sodium ion(s) serve as a driving force for the translocation of the substrate against its electro-
chemical gradient; second, the ions coordinate the binding of the substrate to the transporter; and
third, the ions might function as conformational guides, ensuring that the transporter undergoes the
proper conformational changes during the translocation cycle.
SLC6 transporters bind and translocate one to three sodium ions during the translocation of one
substrate molecule. In addition, several of the transporters bind and cotransport one chloride ion during
one cycle, although this is not a ubiquitous feature of all transporters in the family. The SERT appears
to be special because it also mediates the countertransport of one potassium ion during one transport
cycle. The high-resolution structure of LeuT
Aa
provided for the i rst time insight into the possible
localization of the sodium-binding sites in SLC6 transporters by showing two distinct sodium-binding
sites adjacent to the substrate-binding site. The two sodium ions appeared to have a key role in stabiliz-
ing the LeuT
Aa
core, the unwound structures of TM1 and TM6, and the bound leucine molecule. One
sodium ion (designated Na1) was found to possess an octahedral coordination, with one coordinate
to the carboxyl group of leucine, thereby providing a possible structural link for the coupling of Na
+
and solute l uxes. The other sodium ion (Na2) is positioned between the TM1 unwound region and
TM8, about 7.0 Å from Na1 and not directly involved in coordinating the bound leucine (Figure 14.3).
Notably, with the conservative substitution of a serine for a threonine, all residues coordinating both
Na1 and Na2 are conserved from LeuT
Aa
to the mammalian transporters.
The LeuT
Aa
does not possess any apparent Cl
−
-
binding site, and accordingly the transport of
leucine is not dependent on the presence of Cl
−
. However, the use of homology modeling and energy
minimization of the GAT-1 based on the LeuT
Aa
structure, a potential chloride-binding site in this
transporter, was elegantly identii ed. A cavity in the GAT-1 was found where the chloride ion may
interact with the hydrogen atoms of the amide group of Gln291 and of the hydroxyl groups of
Ser331, Ser295, and Tyr86 (GAT-1 numbering). The model was experimentally verii ed in part by
the introduction of a negatively charged amino acid in position 331 (S331D/E), rendering both the
net l ux and the exchange of GABA largely chloride independent. Equivalent mutations introduced
in the mouse GABA transporter-4 and the DAT also result in a chloride-independent transport,
whereas the reciprocal mutations in LeuT
Aa
and in Tyt1 convey these transporters from displaying
chloride-independent substrate binding to chloride-dependent binding. Furthermore, the transport
rate of GABA increased by lowering the intracellular pH, and thereby likely increasing the pro-
tonation during the return step of the glutamate inserted in position 331 of the GAT-1. This result
suggests that in the wild-type transporter, the chloride ion is a substrate for the GAT and is released
to the cytosol in contrast to simply binding to the protein throughout the entire translocation cycle.
The requirement of the negative charge during the translocation of GABA, but not during the return
step, suggests that the role of chloride is mainly to compensate for the multiple positive charges that
enable accumulation of the substrate against huge concentration gradients.
