(Received for publication, July 21, 1995; and in revised form, October 10, 1995)
From the
Transport of 4-aminobutyrate into Escherichia coli is
catalyzed by gab permease (GabP). Although published studies
show that GabP is relatively specific, recognizing the common
-amino acids with low affinity, recent work from this laboratory
indicates that a number of synthetic compounds are high affinity
transport inhibitors (50% inhibition at 5-100 µM).
Here we present evidence that many of these structurally heterogeneous
compounds not only inhibit transport but also function as alternative
GabP substrates (i.e. a set of observations inconsistent with
the idea that the core of the GabP transport channel exhibits rigid
structural specificity for the native substrate, 4-aminobutyrate).
The gab gene cluster is required for metabolism of
4-aminobutyrate in Escherichia coli. The cluster consists of a
regulatory gene, gabC, two structural genes (gabD and gabT) encoding the metabolic enzymes, succinic semialdehyde
dehydrogenase, and glutamate:succinic semialdehyde transaminase, and a
third structural gene (gabP), encoding the 4-aminobutyrate
transporter (gab permease or GabP). The GabP is a hydrophobic,
466-residue polypeptide (7) that is readily modeled as a
transmembrane protein consisting of 12 transmembrane -helical
segments. The permease is active in whole cells as well as in
rightside-out vesicles (3) , and uptake of 4-aminobutyrate is
stimulated by membrane potential and abolished by proton
ionophores(7) . Recently, we showed that a number of synthetic
compounds are potent GabP inhibitors(8, 9) . An
unanswered question is whether any of these inhibitors might in fact be
transported substrates of GabP. Here, we provide evidence consistent
with the hypothesis that GabP transports at least nine different
substrate analogs.
It is shown directly with radiolabeled compounds
that two bulky and structurally distinct heterocyclic molecules are
efficiently transported by the 4-aminobutyrate transporter expressed
under control of a lac promoter. Transport of 3-piperidine
carboxylic acid or 3-hydroxy-5-aminomethylisoxazole was dependent upon
either the presence of plasmid-borne gabP or upon induction of
gene expression with isopropyl--D-thiogalactopyranoside.
Transport of either novel substrate was inhibited by the native
substrate (4-aminobutyrate) over a range of concentrations consistent
with the transport K
.
Transport of several other inhibitors (3-hydroxy-5-aminomethylisoxazole, 4-amino-cis-2-butenoic acid, 3-piperidine carboxylic acid, cis-3-aminocyclohexyl carboxylic acid, 5-aminopentanoic acid, 3-aminopropanoic acid, 3-aminobutenoic acid) could not be demonstrated directly since these were unavailable in radiolabeled form. However, the above named compounds exhibited behavior consistent with the hypothesis that they can serve as counterflow substrates of the 4-aminobutyrate transporter (i.e. under appropriate conditions GabP can translocate these compounds across the membrane). Among the inhibitors tested, GabP exhibited some preference for translocation of compounds that mimic a nonextended conformation of 4-aminobutyrate (modeled by 4-amino-cis-butenoic acid) over those that mimic the extended conformation (modeled by 4-amino-trans-butenoic acid). Regardless, the data indicate that the GabP transport channel can recognize and/or translocate a far more diverse range of chemical structures than previously imagined. It may be possible to exploit this diversity to develop both genetic and biochemical approaches aimed at identifying amino acid residues that affect ligand recognition and translocation.
Transport reactions were initiated by adding 80 µl of washed
cells with rapid vortex mixing to 20 µl of solution containing the
radiolabeled substrate and other additions (conditions indicated in the
figure legends). A metronome was used to reproducibly time short uptake
reactions (2-20 s). Uptake was rapidly quenched by adding 1 ml of
a ``stop solution'' (buffer containing 20 mM
HgCl) to the rapidly mixing reaction vessel. The
``quenched'' reaction was vacuum-filtered. The reaction
vessel was then washed with 1 ml of 100 mM potassium phosphate
(pH 7.0), 5 mM HgCl
, and this was applied to the
same filter. Finally, the filter was washed with 4 ml of buffer
containing 5 mM HgCl
. The filter was dissolved in
scintillation mixture, and the radioactivity (disintegrations per
minute) was calculated by a Beckman LS3801 scintillation spectrometer
using stored quench curves and automatic quench compensation (H number
determination).
Nitrogen-limiting conditions induce in E. coli the
ability to utilize 4-aminobutyrate as a source of carbon and nitrogen (3, 4, 5, 6) . This capability
derives jointly from induction of the requisite metabolic enzymes as
well as from induction of a permease that catalyzes accumulation of
4-aminobutyrate from the environment. Previous studies (3, 7) agree that the GabP exhibits biological
specificity, rejecting common constituents from the cellular milieu (e.g. the 20 common -amino acids, ornithine, putrescine)
that exhibit at least superficial structural similarity to
4-aminobutyrate.
On the other hand, studies (8, 9) show that a number of synthetic compounds (e.g.Fig. 1) are potent GabP inhibitors. Although these molecules bear some structural resemblance to the native substrate, 4-aminobutyrate (compound 4), the degree of dissimilarity was sufficient to warrant speculation that some of these molecules (particularly the heterocycles) might act at an allosteric inhibitory site rather than within the GabP transport channel itself. The experimental evidence presented here fails to support this hypothesis for at least nine such inhibitors.
Figure 1:
Actions of selected test compounds on
[H]GABA uptake mediated by GabP. E. coli SK55 cells were exposed for 30 s to 10 µM [
H]GABA plus the indicated concentrations of
either compound 1 (
), compound 6 (
), compound 11
(
), or compound 18 (
), at which time the uptake reaction
was quenched with a stop solution containing 20 mM
HgCl
. The cells were harvested by vacuum filtration and
processed for scintillation counting as described under
``Experimental Procedures.'' Control experiments indicated
that compounds 1 (10 mM) and 6 (1 mM) had no
inhibitory effect on the LacY-mediated transport of
[
C]methyl-
-D-thiogalactopyranoside,
suggesting that the observed inhibition of
[
H]GABA transport by these compounds could not be
explained by a global effect on membrane energization (data not
shown).
Several lines of
evidence suggest that both [H]nipecotic acid
(compound 7) and [
H]muscimol (compound 3) are transported quite specifically by GabP ( Fig. 2and
3, respectively). Transport of these compounds depended strongly upon
either the presence of plasmid-borne gabP (Fig. 2) or
upon specific induction of gabP expression by IPTG (Fig. 3). Transport of [
H]GABA, the native
substrate, was likewise induced by IPTG in this expression
system(8) . Moreover, 10 µM GABA was found to
inhibit transport of either [
H]nipecotic acid (Fig. 2, inset) or [
H]muscimol (Fig. 3, inset) by about 50%, an effect consistent with
the published K
(10-12 µM) of
the GabP for GABA(3, 7) . The observed transport of
[
H]nipecotic acid and
[
H]muscimol supports the proposal that these
heterocyclic compounds occupy the GabP transport channel (rather than
an allosteric site) to inhibit [
H]GABA uptake.
Figure 2:
The gabP dependence of nipecotic acid uptake by E. coli K-12. The cells were grown in rich medium supplemented with 0.6
mM IPTG to induce gabP expression. E. coli SK55 (,
) or SK45 (
) cells were washed,
resuspended in 100 mM potassium phosphate (pH 7.0), and then
exposed to 100 µM [
H]nipecotic acid
(0.5 µCi/ml) in the presence (open symbols) or absence (solid symbols) of 1 mM unlabeled nipecotic acid
(compound 7). Inset, washed E. coli SK55 cells were
exposed to 100 µM [
H]nipecotic acid
(0.5 µCi/ml) along with the indicated concentrations of GABA for 60
s. The broken line indicating 50% inhibition was calculated
from the uptake velocity measured in the absence of GABA.
[
H]nipecotic acid uptake was rapidly quenched by
the addition of a mercuric chloride stop solution. The cells were
harvested by vacuum filtration, and radioactivity was quantitated as
described (``Experimental
Procedures'').
Figure 3:
The gabP dependence of muscimol
uptake by E. coli K-12. The cells were grown in rich medium
with (solid symbols) or without (open symbols) 1
mM IPTG to induce gabP expression. E. coli SK55 (,
) or SK45 (
) cells were washed,
resuspended in 100 mM potassium phosphate (pH 7.0), and then
exposed to 1 µM [
H]muscimol (0.4
µCi/ml) for the indicated times. [
H]muscimol
(compound 3) uptake was rapidly quenched by the addition of a mercuric
chloride stop solution. The cells were harvested by vacuum filtration,
and radioactivity was quantitated as described (see ``Experimental
Procedures''). Inset, washed E. coli SK55 cells
were exposed for 15 s to 1 µM [
H]muscimol (0.4 µCi/ml) along with the
indicated concentrations of GABA prior to quenching with stop solution
as described above. The broken line indicating 50% inhibition was
calculated from the uptake velocity measured in the absence of
GABA.
Additionally, it will be noted (Fig. 4) that nipecotic acid
(compound 7) contains within its structure the basic elements of
two open-chain amino acids, 5-aminopentanoic acid (compound 4)
and 3-aminopropanoic acid (compound 10). Thus, nipecotic acid
has the potential to be recognized by GabP in the context of either
compound 4 or compound 10 (or both). Indeed, compounds 4 and 10 were found to inhibit
[H]nipecotic acid transport (Fig. 5) with
IC
values of 10 µM and 200 µM,
respectively. These experiments are in accord with previous studies
showing that 5-aminopentanoic acid and 3-aminopropanoic acid inhibit
[
H]GABA transport with similar apparent
affinities(8) . A related and very important question is
whether compounds 4 and 10 might in fact be transported
GabP substrates.
Figure 4:
Structure of saturated 4-aminobutyrate
analogs. Compound 2 is GABA, the native GabP substrate; it is available
as [H]GABA so that transport has been directly
demonstrated. Likewise, compound 7 is available as
[
H]nipecotic acid so that transport has been
directly demonstrated ( Fig. 2and Fig. 5). Compounds 8,
4, 10, and 9 are not available in labeled form, but transport has be
inferred from the participation of these molecules in the counterflow
reaction (Fig. 7). It will be noted that compound 2 is
incorporated into the compound 8 ring structure. Likewise, compounds 4
and 10 are incorporated into the compound 7 ring structure. In addition
to participating in counterflow, all of these molecules inhibit either
[
H]GABA or [
H]nipecotic
acid transport mediated by GabP.
Figure 5:
Inhibition of nipecotic acid uptake by
open chain amino acids. E. coli SK55 cells were grown in LB
medium containing 1 mM IPTG to induce gabP expression. The washed cells (in 100 mM potassium
phosphate, pH 7.0) were exposed to 1 µM [H]nipecotic acid (0.5 µCi/ml) plus the
indicated concentrations of either compound 4 (5-aminopentanoic acid)
(
), or compound 10 (3-aminopropanoic acid) (
). After 30 s,
uptake was rapidly quenched by the addition of a mercuric chloride stop
solution. The cells were harvested by vacuum filtration, and
radioactivity was quantitated as described under ``Experimental
Procedures.''
Figure 7:
Summary of Inhibitory potency and
counterflow of GabP ligands. Dose-response curves were generated to
assess the ability of each numbered compound to inhibit GabP-mediated
transport in E. coli SK55. The inhibitory potency of compound
2, the native substrate, was assessed using 100 µM
[H]nipecotic acid as the substrate (compound 7).
The remaining dose-response curves were generated to assess the ability
of different compounds to inhibit transport of 10 µM [
H]GABA. These data have been arranged
according to the inhibitor IC
value, which is defined as
the inhibitor concentration that reduces transport to 50% of the
noninhibited control. Compounds represented by white bars behaved as counterflow substrates. Approximate peak values for
counterflow GABA uptake ratio (defined under ``Experimental
Procedures'') were as follows: compound 2, 14-fold; compound 3,
14-fold; compound 4, 30-fold; compound 5, 22-fold; compound 7, 10-fold;
compound 8, 30-fold; compound 9, 15-fold; compound 10, 3-fold; compound
11, 16-fold. In similar studies, no counterflow evidence was obtained
to support the hypothesis that the remaining compounds represented by dark bars are transportable by GabP. Chemical names of
compounds are given in Footnote 1.
Compounds 4 and 10, like most of the
inhibitory compounds described here and elsewhere(8) , are
unavailable in labeled form. Thus, an indirect method, entrance
counterflow(10, 11, 12, 13, 14) ,
was used to determine whether GabP might transport these
nonradiolabeled inhibitors. First, the use of counterflow in this
context was validated by showing that known GabP substrates support
transient [H]GABA accumulation (counterflow) in
metabolically poisoned SK55 cells (Fig. 6). Then the capacity of
many other nonradiolabeled transport inhibitors to support
GabP-mediated counterflow was investigated (see Fig. 7for
summary).
Figure 6:
GabP-dependent counterflow of
[H]GABA driven by nonradiolabeled substrates. E. coli SK55 (
,
,
,
) or SK45
(
) cells were poisoned with 30 mM sodium azide to
prevent active substrate accumulation (see ``Experimental
Procedures''). The poisoned cells were incubated with (solid
symbols) or without (open symbols) an unlabeled compound
(10 mM) in order to assess the effect of preloading the cells
with known (see Fig. 2and Fig. 3) substrates.
Counterflow was initiated by diluting the poisoned cells 200-fold into
medium containing 10 µM [
H]GABA (0.2
µCi/ml). Note that the chemical concentration of extracellular GABA
was 60 µM when GABA was the preloaded substrate. The
preloaded compound was the native substrate (compound 2;
),
3-piperidinecarboxylic acid (compound 7;
) or
3-hydroxy-5-aminomethylisoxazole (compound 3;
).
Within this series of unsaturated test compounds, those that failed to support counterflow share a common structural element (Fig. 8). The carbonyl carbon and the amine moiety are in the trans configuration relative to the double bond that these analogs share in common. It will be noted that the native substrate, 4-aminobutyrate (compound 2), rotates freely about the C2-C3 bond so that the molecule can assume conformations similar to either the cis or the trans isomers of 4-aminobutenoic acid. The profound functional differences between these conformationally restricted cis and trans isomers may imply something about the conformation(s) of GABA that are preferred by GabP.
Figure 8:
Structure of unsaturated 4-aminobutyrate
analogs. Compound 2 is 4-aminobutyrate (GABA), the native GabP
substrate. Free rotation about the C2-C3 bond enables GABA to
assume conformations similar to either the cis (compound 11, a
counterflow substrate) or the trans (compound 18) isomers of
4-aminobutenoic acid. The structure of compound 18 (a nonsubstrate) is
apparent (shaded) in the structures of several other
molecules, compounds 1, 6, and 14(9) , which likewise failed to
behave as counterflow substrates. Interestingly, two of these
molecules, compounds 1 and 6, were rather potent inhibitors of
[H]GABA transport (Fig. 7). Compound 3 is
a relatively potent inhibitor and behaves as a substrate in both direct
uptake assays (Fig. 3) and in counterflow (Fig. 6).
Indeed, with appropriate bond
rotations, the electronegative atoms in 4-amino-cis-butenoic
acid (compound 11) can be made nearly isosteric with those of
another conformationally restricted GABA analog, cis-3-aminocyclohexyl carboxylic acid (Fig. 4, compound 8) so that either molecule might be imagined to dock (hydrogen
bond) with the same complementary surface (e.g. GabP). ()Like compound 11 (4-amino-cis-butenoic
acid), compound 8 (cis-3-aminocyclohexyl carboxylic
acid) is not only a GabP inhibitor but also a counterflow substrate (Fig. 7). When preloaded into metabolically poisoned E. coli SK55, compound 8 stimulated a transient accumulation of
[
H]GABA, which was not observed in non-preloaded
cells. It will be noted (Fig. 4) that part of the compound 8 ring structure indeed mimics the native substrate, GABA (compound 2), which is a high affinity ligand (Fig. 2). The
remainder of the compound 8 ring mimics 6-aminohexanoic acid
(compound 12), a relatively nonpotent inhibitor (Fig. 7)
of GabP. We found no evidence that compound 12 could drive
[
H]GABA counterflow.
Other nonradiolabeled
compounds of interest are 5-aminopentanoic acid (compound 4) and
3-aminopropanoic acid (compound 10). As noted compound 4,
a high-affinity GabP inhibitor (Fig. 5), can be recognized
within the ring structure (Fig. 4) of the transported substrate,
nipecotic acid (compound 7). The remainder of the nipecotic acid
ring mimics compound 10, a moderately potent (Fig. 5)
GabP inhibitor. Preloading of either compound 4 or compound 10 into metabolically poisoned SK55 cells resulted in transient
accumulation of [H]GABA and trans-stimulation of [
H]GABA uptake (Fig. 9). Thus, both 5-aminopentanoate and 3-aminopropanoate
behave as though they are compounds that GabP can recognize, transport,
and couple to a GABA counterflux.
Figure 9:
Counterflow of noncyclic compounds bearing
structural resemblance to the heterocycle, 3-piperidinecarboxylic acid. E. coli SK55 (, ˆ,
,
) or SK45
(
) cells were poisoned with 30 mM sodium azide to
prevent active substrate accumulation (see ``Experimental
Procedures''). The poisoned cells were incubated with (solid
symbols) or without (open symbols) an unlabeled compound
(10 mM) in order to assess the effect of preloading the cells
with noncyclic analogs of compound 7 (see Fig. 4and Fig. 5). Counterflow was initiated by diluting the poisoned
cells 200-fold into medium containing 10 µM
[
H]GABA (0.2 µCi/ml). The preloaded compounds
were 5-aminopentanoate (compound 4;
), 3-aminopropanoate
(compound 10;
), or 3-aminobutyrate (compound 9;
).
Although modest, the uptake ratio for compound 10 was highly
reproducible.
As mentioned, 5-aminopentanoate (compound 4) and 4-aminobutyrate (compound 2) are more potent GabP inhibitors (Fig. 7) than either the longer (6-aminohexanoate, compound 12) or the shorter (3-aminopropanoate, compound 10) open-chain amino acids(8) . The separation distance between the amino and carboxyl groups could be a possible basis for the different inhibitory potencies. However, the behavior of these compounds in counterflow suggests that the hydrocarbon skeleton is also significant.
Like
3-aminopropanoate (compound 10), 3-aminobutyrate (compound 9) is only a moderately potent GabP inhibitor (Fig. 7).
On the other hand, the extra methyl group in compound 9 preserves the charge separation relative to compound 10 and
improves counterflow substantially (Fig. 9). The observed
transient accumulation of [H]GABA and trans-stimulation of [
H]GABA uptake are
consistent with the notion that both 3-aminobutyrate (compound 9) and 3-aminopropanoate (compound 10) are molecules that
GabP can recognize, transport, and couple to a GABA counterflux. That
GabP effectively recognizes compound 9 (3-aminobutyrate) is
consistent with other evidence (see Fig. 8and Footnote 4) that
the permease prefers analogs that mimic a nonextended conformation of
the native substrate, 4-aminobutyrate.
Irrespective of detailed mechanism, the data presented here support a structure/function model in which numerous molecules having different sizes and shapes are capable of interacting with GabP either as inhibitory ligands or as transported substrates. Perhaps most significantly, the counterflow assay has provided the first evidence that several potent to moderately potent inhibitory compounds, unavailable in radiolabeled form, are likely to be transported substrates capable of traversing the core of the GabP transport channel. On the other hand, the counterflow assay also suggested that some potent inhibitors (compounds 1 and 6) that mimic the extended conformation of 4-aminobutyrate do not appear to be counterflow substrates.
In short, a broad range of structurally and perhaps functionally distinct GabP ligands has been identified. The availability of these ligands may allow successful implementation of strategies to select substrate specificity mutants and/or strategies to permit the synthesis of successful active site probes (affinity labels). Thus, dual approaches (genetic and biochemical) to identifying amino acid residues important in the recognition and/or translocation of substrates now appear more feasible than would have been suggested by previous models, which indicated that GabP might be highly selective and unable to recognize alternative ligands.