©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Ligand Recognition Properties of the Escherichiacoli 4-Aminobutyrate Transporter Encoded by gabP
SPECIFICITY OF Gab PERMEASE FOR HETEROCYCLIC INHIBITORS (*)

(Received for publication, May 5, 1995; and in revised form, June 26, 1995)

Steven C. King(§)(¶) Sandra R. Fleming Casey E. Brechtel

From the Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston, Texas 77555-0641

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

4-aminobutyrate metabolism in Escherichia coli begins with transport across the cytoplasmic membrane via the GabP, which is encoded by gabP. Although GabP is specific and exhibits poor affinity for many cellular constituents such as the alpha-amino acids, the range of compounds recognized with high affinity has yet to be investigated. In order to address this gap in knowledge, we developed a gabP-negative host strain, which permits evaluation of test compounds for inhibitory effects on cloned GabP (expression inducible by isopropyl-1-thio-beta-D-galactopyranoside).

Using this inducible expression system, three structurally distinct categories of high affinity transport inhibitor were identified. The structural dissimilarity of these inhibitors significantly alters our view of ligand recognition by GabP. Any complete model must now account for the observation that inhibition of 4-aminobutyrate transport can be mediated either (i) by open chain analogs of 4-aminobutyrate, (ii) by cyclic amino acid analogs, or (iii) by planar heterocyclic compounds lacking a carboxyl group. Such results do not support a previously sustainable view of GabP that features a restrictive ligand recognition domain, unable to accommodate structures that differ very much from the native substrate, 4-aminobutyrate.


INTRODUCTION

The metabolism of 4-aminobutyrate in Escherichia coli depends upon the gab gene cluster(1) , which includes a regulatory gene, gabC, and three structural genes gabD (succinic semialdehyde dehydrogenase), gabT (glutamate:succinic semialdehyde transaminase), and gabP. The latter encodes the 4-aminobutyrate transporter (GabP), a hydrophobic, 466-residue polypeptide (2) that by hydropathy analysis can be readily envisaged as a membrane protein with 12 transmembrane alpha-helical domains. This putative topology is characteristic of many carrier proteins from the plasma membrane of bacterial and animal cells(3, 4, 5) . The GabP is functional in whole cells and in right-side-out vesicles(6) . Active accumulation of 4-aminobutyrate is stimulated by membrane potential and inhibited by proton ionophores(2) . Previous studies have indicated that GabP exhibits considerable selectivity for 4-aminobutyrate and is able to reject the common alpha-amino acids(2, 6, 7) .

The present study is focused on molecules that GabP can recognize rather than on those it can reject. An array of structurally diverse open chain amino acids and their heterocyclic analogs were shown to recognize the GabP with high affinity. These compounds caused a pattern of inhibition, which suggests that the ligand recognition domain of GabP should not be viewed as a restrictive envelope capable of accommodating only the native substrate, 4-aminobutyrate. Instead, there appears to be sufficient flexibility and/or space to allow larger amino acids, smaller amino acids, and conformationally constrained cyclic analogs to be recognized. Even highly planar heterocyclic structures lacking a carboxyl group can be potent transport inhibitors.


EXPERIMENTAL PROCEDURES

Materials

Oligonucleotides were from Integrated DNA Technologies (Coralville, IA). [^3H]GABA (^1)(31.6 Ci/mmol), [^3H]H(2)O (1 mCi/g), and [^14C]taurine (109 mCi/mmole) were from DuPont NEN. 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. Transport inhibitor compounds were obtained from Sigma or Research Biochemicals (Natick, MA). DNA sequencing was performed with Sequenase from Amersham Corp. 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.



Amplification and Cloning of gabP

Twenty polymerase chain reaction cycles were performed using DNA from E. coli DW1 together with forward (5`-agt tta aac gag agg att cag gat ggg gca atc atc gca ac-3`) and reverse (5`-ctt aat taa cgc gta tta tga acg ggt gtt ttt tgc cac-3`) primers that were designed based on the published DNA sequence of the gab gene cluster(2) . The resulting gabP fragment was phosphorylated with T4 polynucleotide kinase and cloned in the EcoRV restriction site of pBluescript II KS(-) to create plasmid pSCK-GP7. The identity of the cloned PCR product was confirmed by DNA sequencing. An expression construct, pSCK-472A, was created by excising gabP from pSCK-GP7 with XhoI-NotI and cloning the gene in the XhoI-NotI sites of pSCK-380. The latter plasmid is identical to the expression vector, pSE380 (tac promoter), except that the NcoI restriction site (and the start codon included within it) has been removed to allow cloned DNA fragments to be expressed utilizing their own start codon and Shine-Dalgarno sequence(8) .

Construction of Strain SK35

A portion of gabP was deleted from the E. coli DW1 by homologous recombination between the chromosome and pSCK-GP7K, a derivative of pSCK-GP7 in which the StyI-PflMI restriction fragment within gabP was replaced by a gene encoding kanamycin resistance. The pSCK-GP7K plasmid was initially placed in DW1 by transformation to create strain SK25. Subsequently SK25 was cured of pSCK-GP7K by serial passage in LB medium containing kanamycin (30 µg/ml) but not ampicillin. A kanamycin-resistant (but ampicillin-sensitive) strain called SK35 was isolated and found to be highly defective for [^3H]GABA uptake (see Fig. 1).


Figure 1: Effect of gabP inactivation on the time course of [^3H]GABA uptake by Escherichia coli K-12. E. coli DW1 (up triangle, filled, ) and SK35 (bullet, ) were grown on minimal medium with glutamine as the nitrogen source. Washed cells (in 100 mM potassium phosphate (pH 7.0)) were exposed to 10 µM [^3H]GABA (0.25 µCi/ml) with (opensymbols) or without (solidsymbols) 10 mM unlabeled GABA. At the times indicated, uptake was rapidly quenched by the addition of a mercuric chloride stop solution. The cells were harvested by vacuum filtration and radioactivity quantitated as described under ``Experimental Procedures.'' Since some data falling immediately along the base line were identical, certain symbols have been offset slightly for clarity.



Cell Growth

In order to ensure induction of chromosomal gabP, E. coli DW1 and E. coli SK35 were grown in an ammonium-free minimal medium (6% Na(2)HPO(4), 3% KH(2)PO(4), 0.5% NaCl, 0.5% glucose, 20 µg/ml thiamine hydrochloride, 5 mM MgCl(2), 5 mM MgSO(4)), using glutamine (0.4%) for a nitrogen source as previously suggested(6) . The medium was sterilized by ultrafiltration (0.22 µm). Cell growth was monitored with a Klett colorimeter (no. 42 filter). Cells grown overnight (16 h) were diluted 50-fold (Klett 10) in fresh medium and grown for around 3.5 doublings (approximately Klett 130).

LB medium (1% Bacto-tryptone, 0.5% Bacto yeast extract, 1% NaCl) supplemented with ampicillin (150 µg/ml) was used to grow the E. 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 0.6 mM IPTG to induce high level gabP expression. Cells were grown for 3-4 doublings (Klett 130-150).

GABA Transport Studies

[^3H]GABA transport was studied under conditions in which gabP was expressed either from the chromosome (in E. coli strain DW1) or from the lac-inducible plasmid, pSCK-472A, contained in E. coli strain SK55. Log phase cells were always 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.''

Transport reactions were initiated by adding 80 µl of washed cells with rapid vortex mixing to 20 µl of solution containing [^3H]GABA and other additions (conditions indicated in figure legends). A metronome was used to reproducibly time short uptake reactions (2-20 s). Uptake was rapidly quenched by the addition of 1 ml of a ``stop solution'' (buffer containing 20 mM HgCl(2)) 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(2), and this was applied to the same filter. Finally, the filter was washed with 4 ml of buffer containing 5 mM HgCl(2). The filter was dissolved in scintillation fluid and the radioactivity (disintegrations/min) was calculated by a Beckman LS3801 scintillation spectrometer using a stored quench curves and automatic quench compensation (H number determination).

Cytoplasmic Water

Cytoplasmic water was taken as the difference between the total aqueous space, measured with [^3H]H(2)O, and the noncytoplasmic [^14C]taurine space(9, 10) . Labeled cells were separated from the bulk aqueous medium by centrifugation through a silicone oil mixture that 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/min 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).

Transport Inhibition

Numerous compounds (pH adjusted to neutrality) were tested for inhibitory activity against GabP. Washed cells (E. coli SK55) were exposed for 1 min to 10 µM [^3H]GABA and the test compound. The IC for each inhibitor was defined as the concentration causing the initial rate of transport to decrease by 50%. The relationship () between IC and the inhibitor dissociation constant, K, is readily derived(11) .

Since the [^3H]GABA concentration in all experiments (10 µM) was set nearly equal to the published (6) K (12 µM), values for K can be estimated to be about 1.8-fold lower than IC. The concentration-response curves used to estimate IC spanned a range that was at least 100-fold (10-fold above and below the reported IC).


RESULTS

As a prelude to the study of GabP inhibitors, a new expression system consisting of a gabP-negative E. coli host strain and an IPTG-inducible gabP expression vector was developed. The system exhibits a number of desirable properties, including the ability to ascribe the vast majority of [^3H]GABA transport (and the effect of test compounds) to the cloned GabP.

The Expression System

In order to gauge the contribution that gabP makes to [^3H]GABA transport in E. coli K-12, we constructed a strain (SK35) in which a 200-base pair segment from the middle of the gene was replaced by a gene encoding kanamycin resistance. Comparison of strains DW1 (functional gabP) and SK35 indicated that [^3H]GABA (10 µM) uptake is gabP-dependent (Fig. 1). The magnitude of nonspecific [^3H]GABA uptake by DW1 (assessed in the presence of a 1000-fold excess of unlabeled GABA) was minimal and the same as uptake measured in the gabP-deficient SK35 strain. These results suggested (i) that gabP is necessary for specific [^3H]GABA uptake in E. coli K-12 and (ii) that SK35 would be a reasonably good host in which to attempt IPTG-inducible expression of a plasmid-borne gabP.

The utility of SK35 as a host strain for high level expression of gabP was shown by cloning the gene in pSCK380, a vector that includes a tac promoter and a lacIfor effective suppression of the cloned gabP (SK35 host strain is additionally lacI). Placing the cloned gabP into SK35 resulted in a new strain, SK55, in which the initial rate of [^3H]GABA transport could be induced 10-fold by IPTG (Fig. 2). A control experiment performed with strain SK45 (containing the plasmid without the gabP insert) shows that induction of [^3H]GABA transport activity by IPTG requires cloned gabP. Comparison of initial [^3H]GABA (10 µM) transport rates by strains DW1 (0.9 nmol/min/mg of protein) and SK55 (6.0 nmol/min/mg of protein) suggests that gabP is overexpressed by about 6.5-fold following brief exposure to IPTG, a sugar that induces expression from the tac promoter.


Figure 2: lac control of gabP-dependent [^3H]GABA transport. E. coli SK45 (up triangle, filled) and SK55 (bullet, ) were grown in LB medium with (solidsymbols) or without (opensymbols) 0.6 mM IPTG to induce gabP expression. Washed cells (in 100 mM potassium phosphate (pH 7.0)) were exposed to 10 µM [^3H]GABA (0.25 µCi/ml). At the times indicated in the figure, 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.''



Others (Niegemann et al.(2) ) have cloned gabP for expression under control of a tac promoter but were unable to directly demonstrate induction with IPTG. The gene was found to be highly toxic, and the permease was thus studied without induction to high level expression. Moreover, the E. coli strain W3110 used in those studies was a prototrophic (nearly wild-type) derivative of K-12, and presumably contained a wild-type gabP on its chromosome. In contrast, the present study shows that host strain SK35, is highly defective for [^3H]GABA transport compared with the parent strain DW1 (Fig. 1). Thus, [^3H]GABA transport measured in E. coli SK55 can be assigned unambiguously to expression from the plasmid-borne gabP, and this transport activity is quite inducible by IPTG (Fig. 2). The [^3H]GABA transport mediated by the cloned gabP was energy-dependent and exhibited a Kof 10-12 µM (data not shown). Thus, the IPTG-inducible [^3H]GABA transport activity expressed by E. coli SK55 conforms to previous descriptions in the literature(2, 6) . E. coli SK55 was used to characterize ligand recognition by GabP.

Open Chain Inhibitors

A series of open chain aminocarboxylic acids of different lengths were studied. Four analogs were found to be inhibitors of gabP-mediated [^3H]GABA transport (Fig. 3). Within the series, C(4) and C(5) compounds were the most potent transport inhibitors (IC = 10 µM), whereas the C(3) and C(6) compounds were 10-20-fold less potent, suggesting that molecular size and/or separation between the amino and carboxyl groups might play important roles in molecular recognition.


Figure 3: Relationship between length of open chain amino acid and potency for GabP inhibition. The inhibitory potency (IC) of the open chain amino acids, 3-aminopropanoic acid (C3), 4-aminobutyric acid (C4), 5-aminopentanoic acid (C5), and 6-aminohexanoic acid (C6) was measured using E. coli SK55 that had been grown in LB medium containing 0.6 mM IPTG to induce gabP expression. In order to estimate IC, the washed cells (in 100 mM potassium phosphate (pH 7.0)) were exposed to test solutions containing 10 µM [^3H]GABA (0.25 µCi/ml) plus a range of inhibitor concentrations. After 1 min, 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.''



Conformationally Constrained Inhibitors

The native GabP substrate, 4-aminobutyrate, is a flexible molecule in which all bonds may rotate freely. Such rotations are restricted in cyclic substrate analogs. Two conformationally restricted cyclic amino acid analogs were found to be potent inhibitors of [^3H]GABA transport (Fig. 4). Both 3-piperidine carboxylic acid (compound 5) and cis-3-aminocyclohexyl carboxylic acid (compound 2) were recognized with apparent affinities (50 µM) that differed about 4-fold from that for GABA itself. (^2)


Figure 4: Inhibition of GabP by conformationally constrained amino acid analogs. E. coli SK55 were grown in LB medium containing 0.6 mM IPTG to induce gabP expression. The washed cells (in 100 mM potassium phosphate (pH 7.0)) were exposed to 10 µM [^3H]GABA (0.25 µCi/ml) plus the indicated concentrations of either compound 2 () or compound 5 (bullet). After 1 min, 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.''



Planar Inhibitors

Tetrahedral geometry characterizes the carbon skeleton of the native GabP substrate (4-aminobutyrate) as well as all of the above mentioned cyclic and open chain transport inhibitors. Although such molecules share a corrugated carbon skeleton in common, this is not a necessary feature for ligand recognition by GabP. Planar heterocyclic compounds are also capable of inhibiting GABA transport (Fig. 5). The planar molecule, 3-hydroxy-5-aminomethylisoxazole (compound 7), was recognized with an apparent affinity (IC = 10 µM) similar to that for GABA itself, whereas 4,5,6,7-tetrahydroisoxazolo[5,4-c] pyridin-3-ol (compound 8), and 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol (compound 9), were considerably less potent, having IC values of 200 and 2000 µM, respectively.


Figure 5: Inhibition of GabP by planar heterocyclic amino acid analogs. E. coli SK55 were grown in LB medium containing 0.6 mM IPTG to induce gabP expression. The washed cells (in 100 mM potassium phosphate (pH 7.0)) were exposed to 10 µM [^3H]GABA (0.25 µCi/ml) plus the indicated concentrations of either compound 7 (bullet), compound 8 (), or compound 9 (black square). After 1 min, uptake was rapidly quenched by addition of a mercuric chloride stop solution. The cells were harvested by vacuum filtration, and radioactivity was quantitated as described under ``Experimental Procedures.''




DISCUSSION

There is a paucity of basic information on the structural selectivity of the substrate recognition domain(s) of GabP. Published studies indicate (i) that 4-aminobutyrate is recognized (6) with relatively high affinity (12 µM) and (ii) that of the 20 common alpha-amino acids, only threonine (6) and aspartate (2) are weakly competitive (K > 1 mM) with 4-aminobutyrate. These studies were fundamentally important, showing that GabP is specific and able to reject common amino acids from the cellular milieu.

On the other hand, rejected molecules (since they fail to interact with GabP) have provided limited information about the molecular architecture of the ligand recognition domain(s). In contrast, well-recognized ligands (i.e. those having high affinity for GabP) are presumably a good fit to the recognition domain and as such provide information to constrain our view of the permease molecule. Models of GabP structure and function ultimately have to account for the observed spectrum of recognizable ligands. The present study indicates that three structurally distinct ligand categories appear to recognize GabP with high affinity. These results place significant constraints on modeling of GabP structure and function.

Carbon Chain Length

GABA, the native GabP substrate, is a highly charged zwitterion in solution when pH is in the physiological range. Since the charged amino and carboxyl groups at either end of GABA are prominent structural features, it is reasonable a priori to assume that these groups interact with GabP. The 4-carbon structure of GABA places an upper limit on the separation distance between the charged amino and carboxyl moieties. Thus, if the GabP were optimized to recognize an extended conformation of GABA, then a shorter molecule might not be able to interact optimally. On the other hand, the charged groups on a longer multi-carbon analog could in principle be brought arbitrarily close together to satisfy requirements of such an optimized permease, but the volume occupied by the carbon backbone would increase with the addition of each methylene group, eventually limiting molecular recognition. An issue not previously addressed is whether, and to what extent, GabP has the ability to recognize potential substrate molecules that tether the amino and carboxyl groups at different distances on otherwise identical carbon skeletons.

The results (Fig. 3) indicate that both the 4-carbon and the 5-carbon open chain amino acids can be recognized with high affinity (approximately 10 µM), whereas longer or shorter molecules are recognized with lower apparent affinity (IC > 100 µM). The diminished capacity of GabP to recognize the 3-carbon analog is consistent with previous studies, showing broad rejection of the alpha-amino acids(2, 6) in which the amino and carboxyl groups are attached to the same carbon atom. Thus, any complete model of GabP would have to feature (i) ligand recognition domain(s) spacious enough to accommodate amino acids larger than GABA as well as (ii) a mechanism to prevent amino acids smaller than GABA (or with less charge separation than GABA) from being effectively recognized.

The space available within the GabP molecule for recognition of larger amino acids has not previously been characterized. The present study has probed this space, employing cyclic amino acid analogs that are potent inhibitors of [^3H]GABA transport. The cyclic analogs have structures (i) that contain considerable bulk compared with open chain structures and (ii) that lack the rotational freedom inherent in open chain molecules. Despite these important structural differences, our observations (Fig. 4) indicate that both compound 2 and compound 5 are recognized with apparent affinities (50 µM) comparable with that of the native substrate, 4-aminobutyrate (compound 1).^2

It will be noted (Fig. 6) that both compound 1 and 6-aminohexanoate (compound 3) are ``contained'' within compound 2. Thus, the cyclic molecule has the potential to be recognized as an analog of either (or both) of these open chain compounds. If the IC of cyclic compound 2 is described merely as the weighted average of the IC for either open chain compound (no doubt a simplification of the actual situation) it follows algebraically that the 6-aminohexanoate moiety contributes about 60% to the overall affinity, while the 4-aminobutyrate moiety contributes the remaining 40%. This highly simplistic analysis is in general agreement with the notion that the bulky and relatively nonpotent 6-aminohexanoate moiety detracts considerably from, but does not abolish, the inherent affinity (10 µM) of GABA (compound 1) for the permease. A similar analysis can be applied to compound 5 as described below.


Figure 6: Structural comparison of open chain and cyclic amino acid analogs. The native GabP substrate is 4-aminobutyrate or GABA (compound 1). Both open chain analogs (compounds 3, 4, and 6) and cyclic analogs (compounds 2 and 5) are effective inhibitors of [^3H]GABA transport (see Fig. 3and Fig. 4). It will be noticed that each open chain compound is contained within the adjacent cyclic structure. Thus, the cyclic compounds have the potential to be recognized in the context of either (or both) of the open chain inhibitors.



Both the potent (IC = 10 µM) transport inhibitor, 5-aminopentanoate (compound 4), and the less potent (IC = 100 µM) 3-aminopropanoate (compound 6) are ``contained'' within the structure of compound 5. Thus, the cyclic molecule has the potential to be recognized as an analog of either (or both) of these open chain compounds. If the IC (50 µM) of compound 5 is analyzed as the weighted average of the IC values obtained for the respective open chain inhibitors, then compound 6 and compound 4 (having IC values of 100 and 10 µM, respectively (Fig. 3)) must receive algebraic weights of 45 and 55%. In other words, the data are consistent with the idea that the inhibitory potency of compound 5 derives about equally from its 5-aminopentanoate and 3-aminopropanoate moieties.

Regardless of whether these simple comparisons and calculations have mechanistic validity, it is clear that GabP recognizes both open chain and cyclic molecules that are significantly larger than GABA itself. These large amino acid analogs (Fig. 6) display amino and carboxyl groups on a nonplanar carbon skeleton. In contrast, compounds 7-9 (Fig. 7) are highly planar and do not feature a carboxylic acid group. Compound 7, the most potent (10 µM) inhibitor in the planar series, is also the smallest and the most flexible molecule. Decreasing the flexibility and increasing the molecular size via addition of a second ring (compound 8) reduced the potency 20-fold (200 µM). The relative position of polar groups in the polycyclic compounds 8 and 9 was also important since the IC of compound 9 (2000 µM) was increased 10-fold over that for compound 8 (200 µM).


Figure 7: Structural comparison of planar heterocyclic GabP inhibitors. The structures of compound 7, compound 8, and compound 9 share in common the planar isoxazole ring. These molecules also lack the carboxyl group that is common to all of the open chain and cyclic amino acid analogs (Fig. 6).



The results presented above suggest a minimal model (Fig. 8) for GabP in which GABA (compound 1) is a transported substrate that can enter the core of the transport channel where it can become occluded by gating mechanisms that are necessary (12, 13) for energy-driven (1, 2) accumulation. The inhibitory actions of the structurally diverse set of molecules described in this communication suggests additionally that portions of the transport channel may be able to accommodate ligands far bulkier than GABA itself. Although the possibility exists that some of these inhibitors act at an allosteric ligand binding domain physically removed from the transport channel, preliminary results from this laboratory^2 suggest that compounds 5 and 7 are indeed transported substrates (i.e. they necessarily enter the transport channel). Thus, we favor the idea that most, and probably all, of the inhibitory compounds discussed in this study act within or at the mouth of theGabP transport channel.


Figure 8: Model for ligand recognition by GabP. GABA (4-aminobutyrate) is a known substrate (2, 6) that can enter the GabP core to occupy an occluded or gated space. Some mechanism of bidirectional gating is necessary for active substrate accumulation by a carrier mechanism (i.e. carriers cannot behave as water-filled channels in which the substrate has contiguous access to the intracellular and periplasmic spaces). The nature of amino acid side chains (R-groups) lining the transport channel or the access channels is completely unknown, but the zwitterionic nature of the substrate strongly suggests that polar residues will be important. The present study suggests additionally that the working model must reflect properties of GabP that allow [^3H]GABA uptake to be inhibited either (i) by open chain GABA analogs, (ii) by cyclic, conformationally constrained GABA analogs, or (iii) by planar heterocyclic compounds having structures that are less obviously related to GABA than the other categories of inhibitor. Whether any of these inhibitors can act at an allosteric inhibitory site remains to be elucidated, but at present the favored view is that the inhibitors interact with some aspect of the transport channel. Compounds 5 and 7, the novel heterocyclic inhibitors for which allosteric actions might be suspected, interact with the channel since they are bidirectionally transported.^2 Accordingly, compounds 5 and 7 are depicted at the mouth of the GabP transport channel for illustrative purposes and without any implication as to whether or not the permease can bind these molecules simultaneously with 4-aminobutyrate.




FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by National Institutes of Health Grant 5 RO1 NS31029-03 and a United States Army Young Investigator Award DAAH04-94-G-0014.

To whom correspondence should be addressed. Dept. of Physiology and Biophysics, Basic Science Bldg., Rte. F-41, University of Texas Medical Branch, Galveston, TX 77555-0641. Tel.: 409-772-1380; Fax: 409-772-3381; sking{at}beach.utmb.edu.

(^1)
The abbreviations and compound nomenclature used were: GABA, -aminobutyric acid; IPTG, isopropyl-1-thio-beta-D-galactopyranoside; compound 1, 4-aminobutyric acid; compound 2, cis-3-aminocyclohexyl carboxylic acid; compound 3, 6-aminohexanoic acid; compound 4, 5-aminopentanoic acid; compound 5, 3-piperidine carboxylic acid; compound 6, 3-aminopropanoic acid; compound 7, 3-hydroxy-5-aminoethylisoxazole; compound 8, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol; compound 9, 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol.

(^2)
Unpublished data (S. C. King) indicate that bidirectional transport of radiolabeled compounds 5 and 7 is vigorous, energy-dependent, and strictly gabP-dependent (comparison with E. coli SK45 and SK55). The IC of 4-aminobutyrate (assessed essentially as described here) for inhibition of inwardly directed transport of compounds 5 and 7 is about 10 µM, a value comparable with the K for [^3H]GABA uptake (2, 6).


ACKNOWLEDGEMENTS

We thank Suzhen Li and Guichun Wang for expert technical assistance.


REFERENCES

  1. Metzer, E., and Halpern, Y. S. (1990) J. Bacteriol. 172,3250-3256 [Medline] [Order article via Infotrieve]
  2. Niegemann, E., Schulz, A., and Bartsch, K. (1993) Arch. Microbiol. 160,454-460 [CrossRef][Medline] [Order article via Infotrieve]
  3. Baldwin, S. A., and Henderson, P. J. F. (1989) Annu. Rev. Physiol. 51,459-471 [CrossRef][Medline] [Order article via Infotrieve]
  4. Botfield, M. C., Naguchi, K., Tsuchia, T., and Wilson, T. H. (1992) J. Biol. Chem. 267,1818-1822 [Abstract/Free Full Text]
  5. Wright, J. K., Seckler, R., and Overath, P. (1986) Annu. Rev. Biochem. 55,225-248 [CrossRef][Medline] [Order article via Infotrieve]
  6. Kahane, S., Levitz, R., and Halpern, Y. S. (1978) J. Bacteriol. 135,295-299 [Medline] [Order article via Infotrieve]
  7. Metzer, E., Levitz, R., and Halpern, Y. S. (1979) J. Bacteriol. 137,1111-1118 [Medline] [Order article via Infotrieve]
  8. Shine, J., and Dalgarno, L. (1974) Proc. Natl. Acad. Sci. U. S. A. 72,1342-1346
  9. Heller, K. B., and Wilson, T. H. (1981) FEBS Lett. 129,253-284 [CrossRef][Medline] [Order article via Infotrieve]
  10. Wilson, D. M., Putzrath, R. M., and Wilson, T. H. (1981) Biochim. Biophys. Acta 649,377-384 [Medline] [Order article via Infotrieve]
  11. King, S. C., and Wilson, T. H. (1990) J. Biol. Chem. 265,9638-9644 [Abstract/Free Full Text]
  12. Christensen, H. N. (1975) Biological Transport , 2nd Ed., pp. 346-350, W. A. Benjaminc, Inc., London
  13. Olsen, S. G., and Brooker, R. J. (1989) J. Biol. Chem. 264,15982-15987 [Abstract/Free Full Text]
  14. Wilson, D. M., and Wilson, T. H. (1987) Biochim. Biophys. Acta 904,191-200 [Medline] [Order article via Infotrieve]

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