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(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
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 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- 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
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 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
[
Several lines of
evidence suggest that both [
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 (
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 (
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
[
Figure 4:
Structure of saturated 4-aminobutyrate
analogs. Compound 2 is GABA, the native GabP substrate; it is available
as [
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 [
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
[
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 [
Figure 6:
GabP-dependent counterflow of
[
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
[
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). ( 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 [
Figure 9:
Counterflow of noncyclic compounds bearing
structural resemblance to the heterocycle, 3-piperidinecarboxylic acid. E. coli SK55 (
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 [ 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.
Volume 271,
Number 2,
Issue of January 12, 1996 pp. 783-788
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
UPTAKE AND COUNTERFLOW OF STRUCTURALLY DIVERSE MOLECULES (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-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).
-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.
-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.
Materials
[
H]GABA (
)(31.6 Ci/mmol), [H]muscimol (20
Ci/mmol), [H]H
O (1 mCi/g), and
[
C]taurine (109 mCi/mmol) were from DuPont NEN.
The [H]nipecotic acid (28.5 Ci/mmol) was from
Amersham Corp. Plasmid pSE380 was from Invitrogen (San Diego, CA), and
pBluescript II KS(-) was from Stratagene (La Jolla, CA).
Restriction enzymes were from New England Biolabs (Beverley, MA).
Kanamycin GenBlock was from Pharmacia Biotech Inc. Bacteriological
media were Difco brand supplied by Fisher (Pittsburgh, PA). Transport
inhibitor compounds were obtained from Sigma or Research Biochemicals
(Natick, MA). DNA sequencing was performed with Sequenase from
Amersham. Bicinchoninic acid protein assay reagents were from Pierce.
Cellulose acetate filters (0.45 µm) were from Micron Separations,
Inc. (Westboro, MA). Silicone oils were Dow-Corning 510 fluid and 550
fluid. Liquiscint scintillation mixture was from National Diagnostics
(Atlanta, GA). Other chemicals were from usual sources. Bacterial
strains and plasmids are detailed in Table 1.
Culture Conditions
LB medium (1% Bacto tryptone,
0.5% Bacto yeast extract, 1% NaCl) supplemented with ampicillin (150
µg/ml) was used to grow the Escherichia coli strains SK45
and SK55 for use in experiments with cloned gabP. Cells grown
overnight (16 h) were diluted 100-fold into fresh medium (approximately
Klett 10) containing 1 mM IPTG to induce high level gabP expression. Cells were grown for three to four doublings (Klett
130-150 with number 42 blue filter).Transport
[H]GABA or
[H]nipecotic acid transport was studied under
conditions in which gabP was expressed from the lac-inducible plasmid, pSCK-472A, contained in E. coli strain SK55. Log phase cells were processed by washing twice with
100 mM potassium phosphate (pH 7.0). The cell pellet was
resuspended in this buffer using 25-50% of the original culture
volume such that the protein concentration was 1-2 mg/ml; such
cells are referred to hereafter as ``washed cells.'' ) 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).Cell Water
Cytoplasmic water was taken as the
difference between the total aqueous space, measured with
[H]HO, and the non-cytoplasmic
[C]taurine space(1, 2) .
Labeled cells were separated from the bulk aqueous medium by
centrifugation through a silicone oil mixture, which was empirically
adjusted to an appropriate specific gravity (i.e. we mixed 7
parts 550 fluid with 3 parts 510 fluid, and after phase separation the
dense fraction was harvested and used in the experiments).
Disintegrations per minute for each isotope were calculated as
described above. Cells utilized in this study contained about 5 µl
of cytoplasmic water/mg of protein, such that the typical data point
represents about 0.5-1.0 µl of cytoplasmic water and
0.1-0.2 mg of protein. The above measured values are roughly
equivalent to 0.6-1.2 mg of cells (wet weight).Counterflow Studies
Twice washed (100 mM potassium phosphate, pH 7.0) cells (approximately 2000 Klett
ml or 3 mg of protein) were resuspended at room temperature in
5 ml of the same buffer containing 30 mM sodium azide. After
incubation for 5 min at room temperature, the cells were centrifuged at
4 °C and resuspended in 100 µl of buffer having either the same
composition as the supernatant or with this buffer plus the preloaded
substrate (10 mM). The cells were placed on ice until use.
Counterflow was initiated by rapidly diluting 5 µl of the cells
with 1 ml of 100 mM potassium phosphate (pH 7.0) containing 30
mM sodium azide and [H]GABA as detailed
in the figure legends. Counterflow was terminated by quenching with 1
ml of 100 mM HgCl. 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
quantitated as described above.Analysis
The counterflow results have been
expressed as a ``GABA uptake ratio'' to avoid any implication
that the intracellular concentration of [H]GABA
is known in absolute terms. The GABA uptake ratio in poisoned cells is
the [H]GABA uptake achieved by preloaded SK55 cells divided by the average ``equilibration''
level of [H]GABA uptake achieved by non-preloaded SK55 cells. Thus, the GABA uptake ratio for
non-preloaded cells is theoretically unity (and in practice it is close
to unity). The GABA uptake ratio is often less than unity for the SK45
strain since there is no basis (no GabP) for rapid equilibration in
these cells. Values substantially greater than unity require coupling
between efflux of the preloaded compound and influx of
[H]GABA.()
-amino acids, ornithine, putrescine)
that exhibit at least superficial structural similarity to
4-aminobutyrate.
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).
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.
, 
) 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'').
, ) 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.
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.
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.
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.''
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.
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).
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;
).
Counterflow with Known Substrates
Preloading
metabolically poisoned E. coli SK55 with either the native
substrate, GABA (compound 2), or the alternative substrates,
nipecotic acid (compound 7) and muscimol (compound 3),
resulted in transient [H]GABA accumulation, which
was not evident in SK55 cells that were not preloaded or in SK45
control cells lacking a functional GabP. 4-aminobutyrate is, of course,
the best known GabP substrate(3, 7, 8) , and
transport of this molecule is clearly reflected in the counterflow data (Fig. 6). Likewise transport of compounds 3 and 7,
shown here to be alternative GabP substrates ( Fig. 2and Fig. 3), is clearly reflected in the counterflow data (Fig. 6). This well known (10, 12, 13, 14) correlation between
counterflow in metabolically poisoned cells and uptake in metabolically
active cells can thus reasonably ()be used to implicate the
GabP in the transport of other compounds that are not readily available
in radioactive form(9) . The results suggest that effective
inhibitors are not always effective counterflow substrates (Fig. 7).Counterflow with Test Compounds
Test compounds 18, 1, and 6 (all unsaturated) failed to support
counterflow activity. Compound 18,
4-amino-trans-butenoic acid, was neither an inhibitor (Fig. 1), nor a counterflow substrate (Fig. 7), whereas
its cis isomer (compound 11) was both a relatively
potent inhibitor (Fig. 1) and a counterflow substrate (Fig. 7). In contrast, compounds 1 and 6 were
relatively potent inhibitors (Fig. 1) but were not counterflow
substrates (Fig. 7).
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).
)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.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.
, 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.
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.
)-D-thiogalactopyranoside; compound 1,
2-(aminomethyl)-5-hydroxy-4H-pyran-4-one; compound 3,
3-hydroxy-5-aminomethylisoxazole (muscimol); compound 4,
5-aminopentanoic acid; compound 5,
1,2,3,6-tetrahydro-3-pyridinecarboxylic acid; compound 6,
5-(aminomethyl)-3-2H-isothiazalone; compound 7,
3-piperidinecarboxylic acid (nipecotic acid); compound 8, cis-3-aminocyclohexyl carboxylic acid; compound 9,
3-aminobutyric acid; compound 10, 3-aminopropanoic acid;
compound 11, 4-amino-cis-2-butenoic acid; compound 12, 6-aminohexanoic acid; compound 13,
4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol;
compound 14, 1,2,3,6-tetrahydro-4-pyridinecarboxylic acid;
compound 15, 4-piperidinecarboxylic acid; compound 16,
4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol;
compound 17, 3-aminopropylphosphonic acid; compound 18,
4-amino-trans-butenoic acid.)H]GABA. That the unlabeled compound can
participate in the exchange reaction implies that it is a substrate
capable of transmembrane passage via the same carrier (i.e. GabP) that transports
[H]GABA(10, 11, 12, 13, 14) .
The transient nature of intracellular [H]GABA
accumulation suggests additionally that the carrier (i.e. GabP) has the capacity (i) to catalyze net efflux of the preloaded
substrate (reflected in the falling phase) and (ii) to transduce
energy, available in the transmembrane gradient of preloaded substrate,
to drive concentrative uptake of [H]GABA
(reflected in the peak ``GABA uptake ratio'' defined under
``Experimental Procedures'').)H]GABA entering from the outside, i.e. a small factor in the formalism of Wright (15) . Alternatively, rapid degradation of a preloaded
substrate could prevent observation of counterflow. However, it should
be noted in this regard that GABA, clearly metabolized by
GabT(16) , nevertheless survives metabolic degradation long
enough to allow experimental demonstration of counterflow (Fig. 6). Other investigators (3) using thin layer
chromatography to analyze the state of intracellular GABA have also
concluded that there is little degradation over the course of a
transport experiment. A positive counterflow result (and the associated trans-stimulation) is more difficult to explain as an
artifact. Possibly, a nonsubstrate inhibitor molecule might be
metabolized to a substrate capable of trans-stimulating
[
H]GABA uptake. However, we doubt that such a
fortuitous mechanism could occur frequently enough to make it an
attractive artifactual explanation for the present results (Fig. 7) in which nine different compounds were shown to support
counterflow. Certainly this artifactual mechanism is unattractive for
compounds 2, 3, and 7 since direct methods have
shown these to be GabP substrates.)
We gratefully acknowledge the expert technical
assistance provided by Sandra Fleming and Suzhen Li.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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