(Received for publication, August 15, 1995; and in revised form, September 18, 1995)
From the
While the -aminobutyric acid (GABA) transporter GAT1
exclusively transports GABA, GAT2, -3, and -4 also transport
-alanine. Cross-mutations in the external loops IV, V, and VI
among the various GABA transporters were performed by site-directed
mutagenesis. The affinity of GABA transport as well as inhibitor
sensitivity of the modified transporters was analyzed. Kinetic analysis
revealed that a cross-mutation in which loop IV of GAT1 was modified to
resemble GAT4 resulted in increased affinity to GABA from K
= 8.7 to 2.0 µM
without changing the V
. A cross-mutation in loop
VI, which swapped the amino acid sequence of GAT2 for GAT1, decreased
the affinity to GABA (K
, 35
µM). These results suggest that loops IV and VI contribute
to the binding affinity of GABA transporters. A substitution of three
amino acids in loop V of GAT1 by the corresponding sequence of GAT3
resulted in
-alanine sensitivity of its GABA uptake activity.
These three amino acids in loop V seem to participate in the
-alanine binding domain of GAT3. It is suggested that those three
external loops (IV, V, and VI) form a pocket in which the substrate
binds to the GABA transporters.
-Aminobutyric acid (GABA) (
)is a major
inhibitory neurotransmitter in the mammalian brain and is widely
distributed throughout the nervous system(1, 2) .
Termination of GABA neurotransmission is achieved by a rapid
sodium-dependent uptake system(3) . Molecular cloning studies
have resulted in the isolation and characterization of cDNAs encoding
four different GABA transporters, GAT1, GAT2, GAT3, and
GAT4(4, 5, 6, 7, 8, 9) .
These cDNAs are part of a family of neurotransmitter transporters
sharing similar structure and amino acid sequences but are different in
their substrate and pharmacological specificities. Selective and
differential localization of GABA transporters has been shown in
GABAergic neurons and glial
cells(6, 7, 8, 9, 10, 11, 12) ,
and it is apparent that each of the four GABA transporters functions in
different tissues in a variety of biological processes. The main
feature separating GAT1 from the other three GABA transporters is the
substrate specificity. While GAT1 exclusively transports GABA, the
other transporters are also able to transport
-alanine and
taurine(3, 4, 5, 6, 7, 8, 9, 10, 11, 12) .
Consequently,
-alanine and taurine inhibit GABA transport by GAT2,
GAT3, and GAT4 but have no effect on GABA transport by GAT1.
Information from sequence analysis and site-directed mutagenesis may pinpoint regions and specific amino acids that are important for the various functions of the transporters. Site-directed mutagenesis revealed that three residues in the transmembrane helices are essential for the activity or the assembly of GAT1. It was suggested that Arg-69 functions in the binding of negatively charged chloride ions(13) , Trp-222 functions in the binding of substrate in a manner analogous to the proposed function of a tryptophan residue in acetylcholine esterase (14) , and substitution of Trp-230 prevented the proper sorting of the transporter into the plasma membrane(14) . The precise substrate binding domain has not yet been defined. Recently, we and others(6, 7, 8, 9) reported that four GABA transporters have different pharmacological properties and GABA binding affinities. It was suggested that these variabilities in drug sensitivity were caused by the different structures of the substrate binding domain.
It has been proposed that the family of
Na/Cl
neurotransmitter transporters
are composed of 12 transmembrane helices, and their N and C termini are
situated at the cytoplasmic side of the membrane(19) . This
structure contains six external loops of various sizes that are denoted
in Fig. 1A (I-VI). We looked for amino
acid sequences in these putative external loops that were nearly
identical among GAT2, GAT3, and GAT4 and were significantly different
in GAT1. Fig. 1B compares the amino acid sequences in
the external loops of the four GABA transporters (see Fig. 1A for their position). It is apparent that in
these regions the amino acid sequences of GAT1 are quite unique.
Site-directed mutagenesis was used to generate GAT1 transporters in
which the amino acid sequences of these loops were identical to those
of GAT3(7, 9) (Fig. 1B). Each mutant
was analyzed by following its GABA uptake expression in Xenopus oocytes(8, 9) . The cRNA-injected oocytes
accumulated up to 70 times as much GABA as uninjected oocytes, oocytes
injected with water, or oocytes injected with the glycine transporter
cRNA. The expression levels for each mutant of GAT1 were evaluated by
assay of [
H]GABA transport activities. Fig. 2shows the kinetics of GABA uptake by the expressed mutants
of GAT1. We introduced the loop structure of GAT3 into loops III and V
of GAT1 (Fig. 1B). An Eadie-Hofstee plot revealed an
apparent V
= 310 and 190 pmol/oocyte/h
for GABA uptake of loop III and V mutants (Fig. 2, A and C). These substitutions increased the V
value 3-fold over that of the wild-type GAT1.
However, substitutions of GAT1 into the corresponding loops III and V
structure of GAT3 did not significantly affect the K
value. These mutants mimicked the V
value
of GAT3 without changes in GABA binding affinity (Table 1).
Figure 1: Mutations introduced into the external loops (III-VI) of GAT1. Putative external loops of GABA transporters are depicted in a transmembrane model (A). Each loop is marked in Roman numerals I to VI. B, amino acid sequences of the external loops III, IV, V, and VI of the various GABA transporters. Mutations were introduced into the underlined residues of GAT1 and GAT3. Swapped sequences are indicated by the arrows.
Figure 2:
Effect of GABA concentration on GABA
uptake by Xenopus oocytes injected with synthetic mRNA of GAT1
and mutations in the extracellular loops. [H]GABA
uptake into mRNA-injected oocytes was assayed at the indicated GABA
concentrations (8, 9) . Expression levels for each
construct were evaluated by [
H]GABA transport
activity. Eadie-Hofstee analysis is depicted in the inset of
each panel. Panels: A, loop III; B, loop IV: C, loop V; D, loop VI.
Oocytes injected with cRNA containing loop IV mutation of GAT1 took
up GABA by a high affinity mechanism (Fig. 2B). This
mutant had a GAT4-type structure in loop IV (Fig. 1). Kinetic
analysis revealed a K of 2.0 µM,
which was similar to the K
value reported for the
high affinity GAT4 GABA uptake(6, 9) . This K
value was about 4-fold lower than that reported
for GABA uptake by GAT1 (K
= 8.7
µM). The mutation in loop IV did not significantly alter
the V
value from that of GAT1 (77
pmol/oocyte/h). This mutant mimicked the high affinity of GABA binding
of GAT4 without change in V
value (Table 1).
In addition, we exchanged the amino acid sequence
of loop VI of GAT1 with that of GAT2 (Fig. 2D). Kinetic
analysis revealed a K of 35 µM, which
is 9-fold higher than the value of GAT1. The mutant showed low affinity
GABA uptake, which was similar to the uptake by GAT2 (K
= 79 µM)(8) . The mutation in loop VI
increased the V
value more than 2-fold in
comparison with that of GAT1. This mutant mimicked the low binding
affinity and the V
value of GABA uptake by GAT2 (Table 1).
-Alanine inhibits the GABA uptake by the
expressed GAT3 and GAT4, and to a much lesser extent by
GAT2(9) . However, GAT1 is not sensitive to
-alanine.
These differences in
-alanine sensitivities prompted us to search
for the location of the
-alanine binding site. Fig. 3shows
the different
-alanine sensitivities to GABA uptake by GAT1, GAT3,
and cross-mutations in their loops. As reported previously,
-alanine did not compete with GABA uptake by GAT1; however, GABA
uptake by GAT3 was sensitive to
-alanine. When amino acids in loop
V of GAT1 were substituted to give identical amino acid sequence as in
the corresponding loop of GAT3,
-alanine inhibited the GABA uptake
activity of the mutated transporter. When similar substitutions were
done in loops III, IV, and VI of GAT1,
-alanine did not compete
with GABA uptake into the oocytes. Strikingly, only three amino acid
substitutions in loop V resulted in
-alanine sensitivity of GABA
uptake. These three amino acid substitutions rendered loop V of GAT1
identical to that of GAT3 (Fig. 1, V).
Figure 3:
The effect of -alanine on GABA
transport by GAT1, GAT3, and cross-mutants in their external loops.
GABA transport was assayed in the absence or presence of 200 µM cold
-alanine during the transport reaction. 5-10
oocytes were used for each experimental point, and the data are
expressed as the average uptake as pmol/h/oocyte. Vertical bars give the standard deviation. The results are given as percentage
of the control activity (100%) without
-alanine by GAT1 and
cross-mutants (A) and by GAT3 and cross-mutants (B).
Mutations introduced into each external loop are shown in Fig. 1B.
To determine
whether the three amino acids in loop V of GAT3 were important for
-alanine binding, we substituted the three residues of GAT3 to the
corresponding amino acids in GAT1. This reverse mutation of GAT3
exhibited less sensitivity of its GABA uptake to
-alanine. This
result suggested that three amino acids in loop V participated in the
-alanine binding domain of GAT3.
The pharmacology of the
mutated GABA transporters was assayed by following the effect of
selective inhibitors of GABA uptake by the various transporters. We
tested five drugs for their ability to block the GABA accumulation by
the expressed mutant transporters. As shown in Table 2, betaine
inhibited the GABA transport by the expressed loop VI mutant of GAT1.
GABA transport by the loop IV mutant of GAT1 was more sensitive to
-GPA and dansylarginine-N-(3-ethyl-1,5-pentanediyl)amine
than that of loops III, V, or VI mutants. However, loop III, IV, and V
mutants retained the original sensitivity to nipecotic acid. The mutant
in loop VI was less sensitive to the drug. It is apparent that
substrate and inhibitor specificities of the GABA transporters were
altered by introducing mutations into their external loops.
An
important unanswered question for the mechanism of transport across the
biological membrane is the location of the transporter's
substrate binding site. An extensive study of bacterial and mammalian
sugar transporters failed to situate the precise location of the
substrate binding site in these
transporters(20, 21, 22) . Several reactive
amino acids such as cysteines were proposed to participate in this
activity, but further studies using site-directed mutagenesis revealed
that these residues are not directly involved in transport or substrate
binding (20, 21) . Extended external loops and
functional groups within the transmembrane helices were formally
analyzed as potential substrate binding sites(22) . Although it
may be that both of these structures are involved in substrate binding
and transport, our work points to the possibility that the short
external loops are involved in primary substrate binding. The fact that
a reciprocal effect was observed in two different GABA transporters
endorses the possibility that indeed loop V in the GABA transporters is
involved in the substrate binding. When three amino acids were
substituted in this region of GAT1, which exclusively transports GABA,
inhibition by -alanine was observed. Conversely, when the same
three amino acid residues found in GAT1 were substituted in GAT3, the
inhibition of GABA uptake by
-alanine was decreased. This
reciprocal effect suggests that indeed this short loop takes part in
the substrate binding site of
-alanine. Although it is almost
certain that other parts of the transporters are involved in this
binding, we think that we positively identified at least one of the
moieties that is involved in this binding. Substitutions in the two
adjacent loops (IV and VI) affected the K
and V
of the modified transporters as well as caused
alteration in their inhibitor specificity. Taking this observation
together with the influence on
-alanine binding by loop V
substitutions, we suggest that these three external loops form a pocket
on the transporter into which the substrate binds. Recent studies with
monoamine transporters using chimeric transporters also concluded that
this region is involved in substrate binding(23, 24) .
It is likely that the location identified in this work is only the
initial binding site of the substrate, and upon binding to this
structure the substrate may move to a secondary site that is more
involved in the transport process. The conclusion reached in the
present investigation should be rigorously substantiated by other
approaches. In the absence of x-ray crystallographic data on any
membrane transporter, the immediate techniques that can supplement this
work are the use of biophysical as well as genetic means of second-site
suppressors. We presently are expressing the neurotransmitter
transporters in bacteria and yeast to provide a ready system for
obtaining second-site suppressors for inactive mutations.