Neurochemical Research, VoL 17, No. 5, 1992, pp. 509-528
The Ligand Binding Site of the Synaptosomal Choline Transporter: A Provisional Model Based on Inhibition Studies* E u g e n e R o b e r t s 1,3 and M a s a o T a m a r u z
(Accepted October 3L 1991)
A topographic model of the ligand binding site of the choline transporter was deduced from inhibition studies with the help of CPK molecular models. It is posited that there are two identical or closely similar hydrophilic anionic sites separated from each other by an hinged, essentially planar but conformationally flexible cationic hydmphobic domain. Subsequently to attachment of external choline to either one of the anionic sites, both sites cooperate in enveloping the ligand by a Venus fly-trap mechanism. This leads to rapid conflgurational changes by which the closedliganded form of the transporter opens up to the interior to release the bound choline. Intracellular K+, a ligand for the choline-binding site, is proposed to be countertransportedby a reversal of the above mechanism. KEY WORDS: Cholinetransport; ligandbindingsite; inhibition;structure-activityrelationships;model.
1. INTRODUCTION
mixing techniques, complete sequence determination, high-resolution crystallographic characterization of relevant constituents, and detailed analysis of their associa(ions and interactions under physiological conditions. Until such data become available, we have sought provisionally to characterize aspects of the choline-binding site or sites of the choline transporter through structureactivity studies with substances that inhibit choline uptake into mouse brain subcellular particles (synaptosomes) (9). Quantitative data obtained with approximately 90 compounds employed in construction of the current model were reported previously, as were details of methods, sources of compounds, and extensive references to pertinent literature (9). These will not all be reiterated herein in the interest of brevity and the maintenance of focus of attention on the model to be presented. Our views have been influenced considerably by the remarkable high-resolution crystallographic analyses of bilobulate periplasmic binding proteins of bacterial transport systems of Quiocho and associates (see ref. 10 and references cited therein) and by kinetic studies of
The sodium-dependent high affinity choline transport system in brain has been studied extensively largely because it is believed to be normally rate-limiting for synthesis of acetylcholine, an important neurotransmitter (1,2). Like other such transport systems, it probably consists of several molecular entities. Required for its activity is at least one choline-recognizing protein that may exist in phosphorylated and dephosphorylated forms (3), a Na + and C1--binding entity (4-7) and possibly ubiquitin (8). Associated lipidic membrane components probably play critically important regulatory roles. Deep knowledge of the mechanism of action of the transporter will require kinetic measurements by stopped-flow rapidi Department of Neurobiochemistry,BeckmanResearch Instituteof the Cityof Hope, Duarte, California91010. z Departmentof Physiology,FujitaHealthUniversitySchoolof Medicine, Toyoake,Aichi 470-11, Japan. 3 To whomreprint requests shouldbe addressed. * Special issue dedicatedto Dr. MorrisH. Aprison. 509
0364-3190/92/0500-0509506.50/0 9 1992 Plenum Publishing Corporation
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Roberts and Tamaru
choline transport in erythrocytes by Deves and Krupka (11-14). Throughout, quantitative results will be presented in terms of ICho values (ICso = concentration of substance 0xM) required to inhibit 50% of the choline uptake with synaptosomes in medium containing 1 IzM choline).
2. STATEMENT OF THE MODEL At the outset, we present our presumptive, necessarily crude topographic model of the ligand binding site (Figure 1) deduced from the data to be considered subsequently with the help of CPK molecular models. There are posited to be two identical or closely similar ovoid
hydrophilic anionic sites, referred to as (x pockets or c~ sites, approximately 8 /~ long and 13 /~ wide (Figure 1A (perspective view) and 1B (top view)), that are separated from each other by a conformationally flexible cationic hydrophobic domain that can become essentially planar (stippled region, Figure 1B, approximately 10 .~ long and 13 /~ wide). We suggest that the latter, designated the 13 site, is "'hinged" in such a manner that the ligand, once attached to one of the o~ sites, is enveloped in "'Venus fly-trap" fashion (15) by the transporter (Figure 1C and 1D) prior to the transporter undergoing subsequent conformational changes required for internalization of the choline to take place (Figure 1C and 113).
3. MAKING THE CONTACT AND TRIGGERING TRANSPORT
.A +
B
I,
I-
8~,
,.I -26 .~
C
D // I
k
'
",~ I
Fig. 1. Topographic models of proposed choline binding site of the synaptosomal choline transporter (see text for description).
The synaptosomal membranes in which the choline transport units are imbedded are negatively charged, with an isoelectric point in the neighborhood of 4 (16-18). These negative charges may be largely contributed by chondroitin sulfate and hyaluronic acid that are electrostatically associated with membrane proteins rather than by intrinsic sialic acid residues (17). The resulting electric field induces dipoles in non-charged groups of membrane proteins and other constituents and also orients the permanent dipole moments of water motecuIes so that they react electrostatically with the membrane surface, forming a negatively charged water layer over it. The ~x sites are presumed to have a higher negative charge density than the membrane surface as a whole, the charges of the cx sites resulting from the presence of negatively charged protein residues, e.g., ~/-glutamyl and/or 13-aspartyl groups that furnish the requisite negative charge at physiological pH of 7.3. Let us consider the following proposal for the "homing" mechanism of choline to its binding site. Cationic charge on the quaternary nitrogen atom of choline, distributed over the surfaces of the entire molecule, together with the hydrogen bonding (H-bonding) hydroxyl group assemble over the molecular surface a coat of water that bears a distributed positive charge. The positively charged, hydrated choline molecule, upon contact, associates electrostatically with the negatively charged, hydrated synaptosomal membrane. The respective water layers on the choline and membrane surfaces exert a dielectric (insulating) effect, limiting closeness of approach. Thermal agitation and other asymmetric physical forces result in a rapid rolling movement of the cationic hydrated choline molecule over the anionic
Modeling the Ligand Binding Site of Synaptosomal Choline Transporter membrane surface until its rate of movement is retarded upon contact with the more highly negatively charged choline recognition site (a site). Anchored by H-bond formation through its hydroxyl group as well as by coulombic interaction and favorable steric factors, the choline molecule orients within an c~ site of the transporter in such a manner that the attractive energy between it and transporter becomes great enough to squeeze out intervening water, in this manner minimizing the energy of the system. In other words, the attractive forces between transporter binding site and ligand are so much greater than that of either of them for water that they associate preferentially with each other, releasing bound water in the process. Although net positive charge is a sine qua non for the attachment of a substance to the ligand-binding site of the choline transporter (Table I and Ref. 19), it is unlikely from data to be presented subsequently that coulombic attraction plays a key role in determining the relative efficacies of cationic substances as ligands for the binding site. Local steric and packing effects and the cost in free energy of desolvation probably play determining roles. Substances with structures similar to choline but without proton-donating or proton-accepting groups, like methyl and ethyl trimethylammonium cations (ICso = 30 ~M and 24 txM, respectively), have considerably lower affinities for the attachment site in the pocket than does choline (ICso -- 0.68 IxM), presumably because they tack the anchorage afforded choline by H-bond formation through its hydroxyethyl group. Therefore, the
Table I. Importance of Net Positive Charge in Inhibition of Labeled Choline Uptake 1 Compound Phenyltrimethyl-ammonium Phenyltrimethyl-silane Choline 3,3-Dimethyl-l-butanol Choline phosphate Betaine Carnitine
Structure + (CH3)3N(CH3)3Si+ (CH3)3NCH2CHzOH (CH3)3CCH2CH20H + (CH3)3NCHaCH20P02 -2 + (CH3)3NCH2CHzCO0+ (CH3)3NCHECH(OH)CO0-
I(;5o (~M) 75 > 10,000 0.68 10,0002 2,9003 > 10,000 > 10,000
i Data taken from reference 9. 2 1,1-Dimethyl-l-ethanol [CH3)3COH] and 2,2-dimethyl-l-propanol [(CH3)3CCHzOH] were similarly ineffective. 3 Value probably too low because of partial hydrolysis to choline during experiment.
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probability of their productive fit into the a pocket is decreased by thermal agitation to a greater extent than that of choline, increasing chances of exit prior to firm attachment. Nonetheless, substances of appropriate size lacking a suitable H-bonding group may be transported by the choline transporter (20,21). Cationic substances larger than choline can fit into an a pocket, which because of its flexibility and size may act metaphorically like a molecular fish net to envelop small cationic substances like tetramethylammonium or choline or to just barely stretch around portions of considerably larger moieties such as quinuclidinium or N-pentyl pyridinium groups (see Figure 4 and related discussion). The hydrophobic 13 region between the e~ sites on the transporter, the stippled region shown on Fig 1, is proposed to be cationic, with its molecular arrangement in the unliganded form tending to give it aspects of an essentially planar surface that possesses sufficient flexibility to undergo mutually shaping interactions with a variety of lipophilic entities. Cationic hydrophilic substances approaching the 13 region would be repelled by it, and energetically speaking, would be propelled toward adjacent anionic ligand-binding c~ sites. In this manner the presence of the 13 region would tend to increase the probability of association of choline or of another hydrophilic cationic ligand with an eL site. From the above, it is predicted that substances that bind to one of the o: sites, to one e~ site and the [3 region, or to both of the a sites and to the [3 region would be competitive inhibitors of choline binding and, therefore, competitive inhibitors of its subsequent transport. Substances that bind only to the 13 domain and not to an site would not compete with choline for its recognition site; but they could prevent configurational changes required for envelopment of choline and transport from taking place and would exhibit non-competitive inhibitory behavior. Both competitive and non-competitive inhibition (mixed inhibition) would be shown by substances, the associations of which are energetically and sterically possible with a sites and the 13 site and with the 13 site alone. It is proposed that association of choline with either one of the a sites is necessary and sufficient for initiation of the series of events involved in transport of choline from outside to the inside of a membrane-bounded volume. Immediately upon binding of choline to one of these sites, configurational changes are posited to ensue that preclude association of additional ligand with the second site. The possibility is entertained that the transporter employs a "Venus fly-trap" mechanism (15) to envelop
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the ligand as a necessary step in the transport process (Figure 1C and 1D). At low concentrations of choline, the probability would be small that simultaneous ligand attachments take place to both sites prior to initiation of configurational changes triggered by the first ligand molecule. We suggest that a doubly liganded transporter unit either does not exist or, if it does, it does not transport choline. As an example, tyrosyl-tRNA synthetase is dimeric and the subunits are arranged symmetrically so that both sites are initially equivalent (22-24). However, the latter enzyme binds only one molecule of tyrosine tightly, the binding of a second one remains undetected even at high concentrations of tyrosine. We suggest that this is also the case with the choline transporter. Although the proposed "rolling search mechanism" of a choline molecule along the membrane for the choline binding site of the transporter might at first appear to be slow and inefficient, high affinity for the site, increase in target size afforded by the presence of two potential binding sites, and repulsion by the adjacent cationic hydrophobic region would be mitigating.
4. CONSIDERATION OF STRUCTURAL DATA PERTINENT TO THE PROPOSED MODEL Understanding of association of ligand and inhibitors with the choline transporter requires detailed consideration of such factors as steric, ionic, and H-bonding interactions and degrees of solvation of both ligands and transporter. Although currently it is not possible to deal quantitatively with these variables, qualitative and semiquantitative approaches are useful. A. Association of Choline with the ~ Site and Closure of the Venus Fly-Trap During Transport. Choline and closely related substances are posited to locate entirely within the ~x pocket of the transporter. The placement of a CPK model of choline (ICso = 0.68 txM) on a planar projection of the presumed pocket is shown in Figure 2A and 2B. That of an equally effective ligand, N-(2-hydroxyethyl) quinuclidinium (ICs0 = 0.64 p~M), is shown in Figure 2C and 2D. In case of the even more effective N-methyl-l-quinuclidinium-3-one (ICso = 0.52 ~zM; Figure 2E and 2F), a ketone group may serve as H-bond acceptor for a proton donating group on the transporter and/or it may bind to an amino group on the transporter by formation of a Schiff's base. Triethylcholine (ICs0 = 2.4 txM) also fits into the pocket and can be oriented to form an H-bond at approximately the same site in the pocket as choline does (Figure 2G). If a Venus fly-trap mechanism such as visualized in Figure 1 were operative, choline and the above two quinuclidinium
Roberts and Tamaru
compounds could be accommodated within it (Figure 2B, 2D and 2F). Triethylcholine is too large, however, to allow closure of the presumptive trap (Figure 2H) and is a weaker inhibitor of choline uptake, possibly because it dissociates readily from the open ~x site, while substances that can be trapped irreversibly within it by closure of the "fly-trap" following attachment do not dissociate once the trap has been closed. Although in our experiments only uptake of choline was tested and not that of the substances tested for inhibition of choline uptake, data of others are consistent with the proposed fly-trap mechanism. For example, tetramethylammonium, imidazoie, ethyl- and propyltrimethylammonium cations enter human erythrocytes (20) and pyrrolcholine and monoethylcholine enter rat forebrain synaptosomes via choline transport systems (2). CPK models of all of the latter substances fit the model of Figure 1 in a manner consistent with the closure of the proposed Venus fly-trap. Orienting the hydroxyl groups in the same manner as for choline in Figure 2A, it can be shown that the methyl, ethyl, and propyl analogs of dimethyl-n-alkyl (2 hydroxyethyl) ammonium cations, substances that are transported by erythrocytes, fit the model appropriately for closure. The butyl analog and higher ones, which are not transported, do not fit. Similarly, a congruence of model fit and transport data was found for the diethyl-n-alkyl (2 hydroxyethyl) ammonium compounds, among which the methyl analog fits and is transported and the higher ones do not fit and are not transported. B. Further Characterization of Topography of the Presumed a Site. A common structural feature is shared by choline and several competitively inhibitory substances that fit entirely within the ~x site (Figure 2) with compounds that possess cationic moieties that fit the a site and have other portions that presumably extend into the [3 region, as is illustrated with the CPK model for n-pentyl-N-[4- (3,4,5-trimethoxyphenyl)-3-butynyl] pyrrolidinium (Figure 3; see also Ref. 25). Side views and views from below are shown in Figure 4A and 4B, respectively, of those portions of choline (ICso = 0.68 ~xM, no. 1), N-methyl-l-quinuclidinium-3-one (IC5o = 0.52; no. 2), 2-hydroxy-ethyl quinuclidinium (IC5o = 0.64 IxM, no. 3), and n-pentylN-[4-(3,4,5-trimethoxyphenyl)-3-butynyl] pyrrolidinium (IC5o = 0.004 txM; no. 4) which are presumed to attach to a specialized ligand recognition site within the ~x site. The ~x site is proposed to consist of a concavity the deeper portion of which is large enough to accommodate within it the eight hydrogen atoms of four consecutive methylene groups (Figures 4A and B, no. 4) or four or six hydrogen atoms from two or three adjacent
Modeling the Ligand Binding Site of Synaptosomal Choline Transporter
513
TOP
Fig. 2. Fit of CPK models of choline and three structurally related substances to the anionic ~x site of the proposed model.
methylene groups, respectively, in the middle region and other appropriate groups at the ends (Figure 4A and 4B, no. 1-3). All of the above-mentioned substances fit such a domain in the manner shown, but the whole of the pocket is posited to be larger than just the above postulated specialized region and to be able to accommodate molecular outcroppings of these substances above the deeper attachment region. C. Substances Whose Constituents Extend Beyond the Confines of the Presumed a Site. Tetramethylam-
monium (Figure 5A; ICso = 30p.M) and tetraethylammonium (Figure 5B; ICso = 24 IxM), both competitive inhibitors of choline uptake (Figure 6B; n = 1 and n = 2, respectively), easily fit into the ~ site. However, the tetrapropyl homolog does not fit the site (Figure 5C), is a much weaker inhibitor (ICso = 260 ~M) than the latter two substances, and reciprocal plots give results typical for mixed inhibition (Figure 6B; n = 3). Although the latter substance does not fit entirely into the proposed a site, it does fit the model if it is assumed to associate
514
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Fig. 3. Fit of the CPK model of n-pentyl-N-[4-(3,4,5-trimethoxyphenyl)-3-butynyl] pyrrolidinium to an e~ site and the 13 domain of the proposed choline binding site.
with both c~ and 13 sites (Figure 5D) in a manner compatible with competitive inhibition and with the 13 site alone (Fig. 5E) non-competitively. Similar modeling results (not shown) were obtained with the tetrabutyl and tetrapentyl homologs. Reciprocal plots for the latter three substances were typical for mixed inhibition (Figure 6B, n = 3 - 5), the proportions of noncompetitive inhibition increasing with increasing sizes of alkyl groups. As the size of the alkyl groups in the above series increases, hydrophobicity increases progressively so that attractive forces for the hydrophobic cationic 13 domain are increased, and the single positive charge is diffused over a greater surface, decreasing repulsive forces. The above changes are reflected in the fall and subsequent rise in inhibitory potencies (ICso values) with progressively increasing chain length (Figure 6A) as the nature of the inhibition shifts from competitive to mixed inhibition and finally to purely non-competitive inhibition (Figure 6B). The tetraheptyl compound just fits onto the proposed hydrophobic 13domain of the suggested model (Figure 5F), while the model of the tetraoctyl compound is too large to fit into it. Examination of naked space-filling models of substances often does not permit estimation of their true dimensions in aqueous solutions. Differing degrees of solvation at various molecular sites may play important roles in determining actual molecular sizes and shapes and, thus, may be decisive in determining biological reactivities. For example, IC5ovalues for tetramethylammonium and trimethylethylammonium are 30 txM and 24 p.M, respectively (Figure 7A). The latter substances have relatively weak associations with the water around them and can relatively readily relinquish their water layer in favor of the more energetically favorable binding
to sterically appropriate regions of the choline transporter. However, the hydroxyethyl group confers an especially high affinity on choline (IC5o = 0.68 IxM), indicating that H-bonding must play a key role in enabling the trimethylammonium portion of the choline to come easily within van der Waals' distance of the architecture of the negatively charged cx site. Some quaternary trimethylammonium substances, shown in Figure 7B, may inhibit choline transport by blocking choline entry into a sites in a plug-like manner. The signatures of such substances are ICso values of approximately 300 ~M (Figure 7B). Attached to the nitrogen atom of the latter are groups which either are proton donors or acceptors. Such groups can H-bond with water molecules, around which, in turn, can be organized clusters of additional H-bonded water, the sizes and shapes of the dusters being determined by various molecular and environmental factors. Such bound water may produce distortions in surface geometry and cause effective increases in regional molecular size. If one imagines the nitrogen atom to be at the center of a tetrahedron, there would be methyl groups in three of the corners and the fourth (hydrophilic) group would be at the fourth corner of the tetrahedron. The cartoon in Figure 7B indicates water bulk around groups other than hydroxyethyl with potential for H-bond formation. We propose that such cationic substances, coulombically attracted to the anionic cx site, are able to insert trimethylammonium portions of the molecule into the neighborhood of the cx site, but that entry of the bulky hydrated regions may not take place beyond the rim of the concavity, thus preventing productive fit of the trimethylammonium into the ~x site. Similarities in ICso values for the substances listed in Figure 7B suggest that,
Modeling the Ligand Binding Site of Synaptosomal Choline Transporter
515
A
1
2 Fig. 4. Side views (A) and views from below (B) of the fit to the ~xsite of CPK models of the following substances: 1, choline; 2, N-methyl-1quinuclidinium-3-one; 3, N-(2-hydroxyethyl) quinuclidinium; and 4, the pyrrolidinium portion of n-pentyl-N-[4-(3,4,5-trimethoxyphenyl)-3-butynyl] pyrrolidinium
3
4
B
I
2
3
4
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Roberts and Tamaru
Fig. 5. Substances that fit the ct site alone (A and B), one ct site and the 13 site (D), or the 13 site alone (F).
as long as the dissimilar fourth (non-methyl) substituents contain H-bonding groups, their exact structure has little effect on the affinity for the choline transporter. As more hydrophilic groups are added, progressive decreases in affinity for the transporter occur. For example, among several pyridinium compounds tested (Figure 8), the most potent inhibitor was N-cetylpyridinium, a quaternary ammonium cation with no possibility of forming H-bonds. The next most effective substance was 2,4,6-trimethylpyridinium with one Hbonding group. Next in order of decreasing affinities were N-methyl nicotinamide, 4-aminopyridinium, and 3,4-diaminopyridinium with 3,4, and 7 H-bonding groups, respectively. Thus, relative inhibitory potencies for choline uptake were inversely correlated with potential for H-bond formation and, therefore, hydration. Steric factors introduced by hydration appear to supersede coulombic forces, since among the above substances 3,4diarninopyridinium with three positive charges, but with potential for forming seven H-bonds, had the lowest affinity.
Alkyl trimethylammonium compounds, from tetramethylammonium to n-hexyltrimethylammonium (Figure 9, no. 1-5), can fit completely into the postulated pocket. In this series of substances, intramolecular interaction is postulated to occur, the non-methyl alkyl groups being folded back upon themselves, as might be expected energetically for hydrophobic moieties in a hydrophilic environment. However, with increase in chain length, the tendency is increased for protrusion of the hydrophobic alkyl group outside of the a site into the adjacent 13 hydrophobic region and of association with it (see Figure 7C). If there were no hydrophobic domain on the transporter adjacent to the anionic a pocket, a protruding alkyl chain would tend to fold back upon itself, giving the most compact sterically permitted structure because of the strong repulsive forces between it and the bulk water surround. However, if the hydrophobic domain on the transporter were to act essentially like a shallow lipidic pool, the alkyl chain would tend to extend from the trimethylammoniummoiety in a manner compatible with maximal hydrophobic association (Fig-
Modeling the Ligand Binding Site of Synaptosomal Choline Transporter
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Fig. 6. A: Log ICao values of saturated straight chain symmetrical tetra~lkylammonium compounds as a function of chain length. B: Double-reciprocal plots of choline uptake as a function of choline concentration in the absence and presence of symmetrical tetraalkylammonium compounds, n indicating the number of carbon atoms per saturated straight chain alkyl group. Typically competitive results were obtained for n = 1 and 2; mixed type of inhibition was shown by n = 3 and 4; and characteristically non-competitive inhibition was shown for n = 5 and 6 (from Ref. 9, with permission).
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ure 9, no. 6-9). PolymethyIene chains would extend linearly into such an hydrophobic environment since intramolecular interaction of chain components would tend to be energetically less favorable than association with a flexible hydrophobic pool, linear extension being limited onIy by dimensions of the latter. Once the Iength of
the chain exceeds the permissible linear dimensions, chain bending would begin to take place (Figure 9, no. 9). When bending is no longer physically possible, energetically less favorable folding of the chain would begin to take place in order for the carbon chain to fit into the lipid domain. With progressively increasing length of
518
Roberts and Tamaru
R
ICso(g.M)
A - CHzCHzOH -CHzCH 3 -CH 3
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carbon chain, forces generated by intramolecular chain interaction, hydrophobic interaction with the lipid domain, and repulsion from the overlying water layer could cause polymethylene chains to curl up on themselves and to pull the quaternary group out of the o~ pocket into the bulk water above the lipid pool, metaphorically resembling the familiar cartoon of a cobra with its body coiled on the ground and its head held in the air. If the latter were the case, inhibition of choline uptake would become non-competitive, since competitive inhibition is presumed to require association with an anionic a site. In the case of n-hexyltrimethylammonium (Figure
9, no. 5a and 5b), there may be a balance between the two types of interaction with the transporter, one with the o~ pocket alone (Figure 9, no. 5a; see also Figure 7a) and the other with both the c~ pocket and the [3 region (Figure 9, no. 5b; see also Figure 7C). Alkyl trimethylammonium cations in which the fourth alkyl group is n-heptyl or longer cannot fit into the c~ pocket and, therefore, probably would assume the above type of dual combination rather than the energetically highly unfavorable configuration in which the hydrophobic alky! group would extend into bulk water. Inhibition of uptake would be competitive with choline because binding of
Modeling the Ligand Binding Site of Synaptosomal Choline Transporter | H3N
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the quaternary head group would be occurring to the o~ pocket, attachment site of the choline ligand. This was, indeed, found to be the case for all such substances up to and including n-dodecyltrimethylammonium (Figure 9, n = 12; Figure 10). Substances with carbon chains longer than 12 were non-competitive inhibitors of choline uptake (Figure 10). Although the positive charges on the trimethylammonium groups of the latter substances may help guide them into the vicinity of the transporter, the purely noncompetitive nature of the inhibition is consistent with the idea that, in cobra-like fashion, the long alkyl chain may associate in tightly coiled fashion solely with the hydrophobic 13 region on the transporter while the trimethylammonium head group extends into the bulk water layer above it. D.A More Detailed Topography of the Presumptive a site. Comparisons were made of inhibitory effects on choline binding of quinucIidinium and trimethylammonium cations (Table II). The probability of locating in the choline-specific steric niche of the c~ site is greater for quinuclidinium than for trimethylammonium. Inhibitory potencies were enhanced greatly by substitution of a methyl group for an H-bonding, solvation-enhancing proton on the nitrogen atom. Quinuclidinium and its Nmethyl derivative were more effective than trimethylammonium and tetramethylammonium cations, respec-
519
tively. Replacement of the proton on the nitrogen of quinuclidinium or trimethylammonium with an hydroxyethyl group not only resulted in remarkable enhancement of inhibitory efficacy but also eliminated differences between quinuclidinium and trimethylammonium compounds. When the H-bonding hydroxyethyl group was present, the quinuclidinium ring structure offered no advantage. In substances with no H-bonding groups, anchorage for a cationic group in a manner that improves its association with the oL site can be furnished by an hydrophobic group that associates with the 13 domain. Similar inhibitory potencies were obtained with quaternary trimethylammonium compounds (Table III) whether the fourth, non-methyl group was a saturated straight chain 12-membered polymethylene chain (dodecyl) (no. 3) or an unsaturated aromatic moiety (3,4,5-trimethoxyphenyl)-3-butynyl) (no. 4). However, neither of the latter substances had as high an affinity as choline, itself (no. 5). The H-bonding hydroxyethyl group of choline obviously plays a key role in fixing the trimethylammonium moiety of choline securely in place within the transporter binding site. By keeping other structural features constant and varying the nature of the cationic group, comparisons may be made of degrees of steric fit of cationic groups within the o~ site, as in the case of several (3,4,5-trimethyoxyphenyl)-3-butynyl and 3-carboxyethyl amines (Table IV; Ref. 9 and 25). The weakest inhibitor, the pyrrolidinium derivative (no. 1) can H-bond and associate with water in size-increasing fashion. The next weakest inhibitor, an aza analog of quinuclidinium (no. 2), also has H-bonding capacity. As the structures of the several quaternary ammonium substances (no. 3-6) progressively approached a rigid cage structure (no. 7), inhibitory potency increased. The most effective substance of the series was the N-pentyl piperidinium compound (no. 8), which was more potent than the N-methyl (no. 5), N-ethyl (no. 6), and N-n octyl (no. 9) compounds, suggesting that it meets size and local packing requirements for fitting into the anionic pocket of the proposed model better than do the other substances. On modeling, the N-pentyl piperidinium moiety fits snugly into the proposed c~ site, almost completely filling it. The Nmethyl and N-ethyl compounds fit more loosely. The Nn-octyl analog only could fit the ~x site if a portion of the n-octyl chain were to extend into the aqueous domain above the oLpocket. Since the latter is energetically unfavorable, a decrement in affinity occurs. In Fig. 3 is shown the fit to both the o~ and 13 sites of the N-pentyl pyrrolidinium derivative, which was as potent as the piperidinium compound (25).
520
Roberts and Tamaru
Fig. 9. Effects of increasing chain length of saturated alkyl groups in quaternary trimethylammonium compounds on association with the proposed choline binding site and on inhibition of choline binding.
Of the four quaternary ring-substituted trimethoxyphenyl carboxyethyl ethylammoniums listed in Table V, the quinuclidinium derivative (no. 4; IC5o = 0.018 ~M) was by far the most potent inhibitor of choline uptake. The aza analogue of the quinuclidinium derivative (no. 3) was much weaker (IC5o, 1.8 IxM), probably because the proton on the tertiary ring nitrogen H-bonds with water, seeding formation of a sterically hindering cluster of water molecules around it. The pyridinium derivative (no. 2; IC5o = 7 ~M) was weaker than the two foregoing substances probably because the repulsive effect of the high electron density of the aromatic ring attenuates the effects of the coulombic attraction between the cationic quaternary nitrogen atom and the anionic c~ site on the transporter. The weakest inhibitor of the series (no. 1; IC5o = 10 IxM) was the N-benzyl piperidinyl derivative in which both aromatic-repulsive and sterically-hindering factors are operative. E. Probing the Putative ~ Domain. The second
quaternary ammonium group of decamethonium (Table VI; no. 3) resulted in only slightly greater inhibition of choline uptake than was observed with n-dodecyltrimethylammonium (no, 2), a monoquatemary substance of the same extended length. A closer spacing of the positive charges, as in hexamethonium (no. 6), decreased inhibitory potency below that of n-octyltrimethylammonium (no. 5), the isolength monoquaternary compound. The above data support the suggestion made previously that the hydrophobic region of the transporter bears a positive charge, since a positive charge on a ligand in that portion of the molecule that associates with the putative 13cationic hydrophobic region would be repelled and would weaken ability of a substance to bind (no. 6). Supporting the latter supposition is the observation that the tertiary ammonium compound promazine (ICs0 = 66 I~M) is a weaker inhibitor of choline binding than chlorpromazine (ICso = 26 ~M) (9), the two differing from each other only by the presence of a
Modeling the Ligand Binding Site of Synaptosomal Choline Transporter
521 5x10-6M
05 I
n:6
2 xlO_SM
,~ n t [ o l / '7
n:8
03 ~
t
5x10-SM
"
n
roI
O) 05 [-
~
n=9
/
2•
03 I
....
r
n=16
02 0.] i
i l
~ f
I
L
I
O3L
I
I
I
n=lO
I
I
5•
031
rol
n=]8
5xlO-5M
01 rol
-&
~ 1
/ /
-04
i
0
I
I
04
I
I
0.8
I
I
10
I
i
-08
~
i
I
-04
0
I
Q4
[
08
I
I
1.2
(1/CHOLINE) [}zM] -1 Fig. 10. Double-reciprocal plots of choline uptake as a function of choline concentration in the absence and presence of alkyltrimethylammonium cations in which the saturated straight chain alkyl group varied from 6 carbons (n = 6) to 18 (n = 18). Compounds with n = 6 to n = 12 were strictly competitive, while n = 14 and above were non-competitive (from Ref. 9, with permission).
chlorine atom in the latter in that portion of the molecule that is presumed to locate at the 13 site of the transporter. A chlorine atom has 3 lone pairs of electrons, which increase the negativity of a compound containing it, thereby increasing its affinity for a cationically charged surface. Replacement with hydrogen atoms of two of the methyl groups of the trimethoxyphenyl group of the 3butynyl amines of N-ethyl pyrrolidinium and N-ethyl piperidinium (Table VII; Ref. 25) produced a similar reduction of inhibitory potency of both substances. This may be attributed to increases from 0 to 2 in number of H-bonding groups (one for each methyl group replaced), the increasing hydrophilicity resulting in decreased attraction for the hydrophobic 13 domain. The effect of
methoxy group replacement with hydroxyl groups is much more drastic in the case of the trimethoxyphenyl carboxyethyl N-ethylpiperidinium compound than for the other two substances. Replacement of only one methyl group with an hydrogen atom results in a much greater decrement in inhibitory potency than removal of two such groups in the case of the butynyl amines. The methoxyphenyl 3-butynyl amines of N-ethyl pyrrolidinium and N-ethyl piperidinium were more potent inhibitors of choline transport than the comparable carboxyethyl Nethyl-piperidinium compounds (Table VII). The high electron density of the triple bond in the former two classes of substances enhanced affinity for the cationic hydrophobic surface of site 13, while the H-bonding of the ester carbonyl with water in the latter decreased af-
522
Roberts and Tamaru
Table II. Comparison of Inhibitory Potencies of Quinuclidinium and Trimethylammonium Compounds Compound
Structure
Quinuclidinium
IC5o (~M)
H
40
~/NH
340
\r Trimethylammonium
@ ~5~N-CH3
N-Methyl quinuclidinium
4.7
\@ Tetramethylammonium
~/N-CH3
N-hydroxyethyl quinuclidinium
@ ~N-CHaCH2OH
30 0.64
\@ Choline
~/N-CH2CH2OH
0.68
Table III. Effect of Nature of the Fourth Substituent on Inhibition of Choline Uptake by Quaternary Trimethylammonium Substances + (CH3)3N-R
No.
R
1
- H
2
- CH3
3
- (CHz)llCH3
4
- CHzCH2Cm C ~
5
- CH2CH20H
IC5o (~M)
OCH3 OCH3 OCH3
Presumed association site on model
340
cx
30
c~
3.4
a + [3
2
c~ + [3
0.68
o~
finity for the hydrophobic surface (see Figure 8 for example of effects of H-bonding groups). In each particular instance an increase in numbers of H-bonding groups was accompanied by increasing ICso values (decreased inhibitory potency).
F. Inhibitory Effects of Bisquaternary Ammonium Compounds: Comparison of Efficacies of Mono- and Bisquaternary Compounds. Hemicholinium-3 (HC-3) is the most potent inhibitor of this particular group of substances (ICso = 0.02 IxM). Considering it to exist in the open chain structure, it possesses exceptionally favorable features for attaching to both of the e~ sites and the /3 domain of the proposed transporter surface (Fig. 11D).
The two not quite coplanar benzene rings span the hydrophobic [3 domain in such a manner as to allow the two choline-like quaternary groups not only to associate with both ot sites with good steric fit but also to form H-bonds through its hydroxyl and ketone groups with protein backbone or side chain constituents. It is important for the quaternary head groups to extend sufficiently beyond the/3 domain in order to allow the mutual shaping to take place between ligand and transporter necessary to achieve optimal steric fit within the o~ sites. The latter point is illustrated further by comparison of inhibitory efficacies of substances in which quaternary ammonium groups are separated from each other by different numbers of methylene groups. Decamethonium with 10 methylene groups (Figure llB) is a much weaker inhibitor (ICso = 1.5 tzM) than similar compounds with 18 methylene groups between two trimethylammonium groups (IC~o = 0.088 IzM) or two triethylammonium groups (IC6o = 0.05 ~M; Fig. llC). Ten methylene groups just barely extend across the hydrophobic domain, and considerable deformation of the et site structure would be required for envelopment of the quaternary groups. In addition to furnishing larger regions for contact with hydrophobic regions of the /3 domain, longer polymethylene chains may allow the quaternary groups to dip freely into the ~x pockets to make the fit in such a manner as to minimize requirement for energetically less favorable configuration of et site structure and of the structures of ligands. In Figure 12 are summarized our previous data (9) on the efficacies of dimethylammonium and trimethylammonium mono-quaternary compounds in inhibiting choline uptake and of bisquaternary alkyl compounds (26). Maximal inhibitory potency of the monoquaternary compounds occurred at carbon chain length of 11 and that of the bisquaternaries at 18. The lower efficacies of monoquaternary compounds than those of the bisquaternaries are consistent with the suggestion of the presence of two anionic sites on the choline transporter separated by a cationic lipophilic domain. The question arises as to the reason for the increasing potency with chain length in the bisquaternary series beyond the chain length of 10, even though the latter probably adequately can span the distance between the two anionic sites. One possibility is that the longer carbon chains can be folded in such a way as to give both greater hydrophobic interaction with the transporter and greater rigidity in keeping apart the two anionic binding sites of the transporter than would a substance with a single extended carbon chain that is just barely long enough to span the distance between the two presumptive anionic domains. Another possibility may be that the peptide chains linking the
Modeling the Ligand Binding Site of Synaptosomal Choline Transporter
523
Table IV. Inhibitionof Choline Uptake by Trimethoxyphenyl-butynyland Trimethoxyphenyl-carboxyethylAmines ,--r" OCH3
9
,~/OCH3
RCH~C~C---CO-~OCH~ t RCH2CH2OC~-OCH3 "OCH3 ]. No.
R Ref. 25
Ref. 9
Ref. 25
0.3
0.2
4
6
0.05
0.18
0.08
7
0.054
0.09
0.018
0.005
0.02
0.02
0.12
0.23
0.25
1 2
10 HN H ~ N -
,/.-
3 4
5 2 0.7
9
Table V. Effectsof QuaternaryRing-SubstitutedTrimethoxyphenyl CarboxyethylEthylammoniums 0
RCH2CH20~ ~
0CH3 OCH3
--"OCH No.
R
1
2 3
4
"OCH3
ICso (I~M)
ICso (IxM)
10
@ o/--~ H-N N-
7 1.8
0.018
two c~ sites of the transporter may be capable of being stretched beyond their minimal-energy length of 10 ~ when
two quaternary groups fitting both c~ sites are separated by longer intervening groups that participate in hydrophobic interactions with the peptidic linkers. This may help explain why in one study (27) it was found that terphenylhemicholinium-3, which contains one phenyl group more than hemicholinium-3 (see Figure 11D), was as potent an inhibitor of choline transport as is hemocholinium-3.
A MODEL FOR CHOLINE TRANSPORT Now we propose to locate the choline binding site in an overall model of choline transport (Figure 13). The two identical oL sites are presumed to be located either in two regions of a single protein or on two identical separate protein subunits (Fig. 13A). The irregular junctional region shown in the center of the 13 domain is meant to represent a non-covalent hydrophobic interaction of molecular extensions from the two e~ sites that are part of the hinge region of the Venus fly-trap mechanism. Such a region would allow the [3 site to be
524
Roberts and Tamaru Table V I . R o l e s o f S i z e a n d C h a r g e I
No.
ICso (IxM)
1
CH3 CH~
+1 - N r
-
4.2 (CH2)~-
CH~
(CH2) 9 -
CH2 -
CH3
CH3
+F 2
CH 3 -
N -
r
CH2 -
CH3
3.4
CH3
CH~
CH3
+l 3
CH 3 -
+1
N -
(CH2)9 -
CH2 -
N
I
I
CH3
CH3
-
CH3
1.5
CH3
+I 4
CH 3 -
N -
(CHJs-
CH3
(CH2)5-
CH z -
70
I CH3
CH3
+1 5
CH 3 -
N -
CH 2 -
CH3
24
I CH3
CH3
CH3
+1 6
C}~3 -
N -
+I (CH2) 9 -
I cg 3
CH z -
N
-
CH3
i30
I CU 3
i D a t a t a k e n f r o m r e f e r e n c e 9.
"stretchable" in special instances, such as when there is attachment of some over-size bisquaternary compounds, as discussed above. It atso might even be temporarily breakable at a particular stage of the conformational changes occurring in the transport sequence (Figure 13C). At any time during transport there would be three active forms of the transporter, open-unliganded (Fig. 13A with no ligand), open-liganded (Figure 13A with ligand attached), and closed-liganded (Figure 13B). In addition, there must be one or more inactive forms of
transporter (Figure 13D) since activation of transport can be achieved in a number of ways. The choline transporter normally may exist kinetically in distribution between these several forms. Lipid-protein or protein-protein interactions at one or more sites of the membrane-located transporter with other constituents may limit the rate at which the transporter can undergo the configurational transitions required for all of the transport steps to take place (Figure 13). These relationships would be expected to be sensitive to a wide variety of chemical and physical influences. There is a well-known dependence of choline uptake on Na + and CI- ions, increasing concentrations of the latter producing progressively increased transport, achieving a maximal effect at 0.15 M Na § the physiological extracellular concentration (9). The synaptosomal choline uptake system we employed is not amenable to extensive studies of effects of ions because of its complexity. Drawing on studies of GABA transport performed with a more comminuted membrane preparation (28), we propose that the choline transporter consists minimally of two entities,s a choline recognition protein and a Na+-dependent protein required for activity of the latter which may be associated with it non-covalently or covalently bound to it. This suggestion is compatible with the finding in E. colt that diverse Na § gradientcoupled transporters share in common a genetically coded subunit which is responsible for the Na + translocation (29). We suggest there to be a cooperative, mutually shaping relationship between the subunits so that the conformational changes brought about by Na + and CIattachment allow the choline recognition site to become functionally active. There is proposed to be a transducer zone cr interface at the conjunction of the subunits at which conformational changes in one subunit may be transduced into conformational changes in the other. Important inhibitory or facilitatory effects could be exerted at one or more points in this region by membrane components (proteic or lipidic) other than the subunits, themselves. For example, association with closely lying membrane phospholipids such as phosphatidyl choline, might serve to restrain the choline transporter from becoming active, the choline head groups possibly dipping into ~xsites. Weakening such an association at the transducer zone of the transporter might increase the rate of choline transport, which normally might occur when the entire choline and ion-loaded transport entity undergoes the conformational changes by which the choline is released to the interior. To summarize, activational influences resulting from attachment of ions and choline to their respective binding sites on subunits of the transporter greatly increase the
Modeling the Ligand Binding Site of Synaptosomal Choline Transporter
525
Table VII. Decrementin Inhibitionwith Increasesin H-BondingGroups* IC~o(~M) I---~|
~
OCH3 H H
R2
Rs
OCH3 OCHa OCHa
OCHa OCHs n
~OR~ I_~,,N-CHzCHzC=C - ~-~.~ ORz CHzCH 3 OR3
~|
/_~OR 1
~-CHzCHzC~C- ~-ORz CHzCH ~
0.02 (0)
0.0S (0)
1.0 (2)
0.9 (2)
'OR~
/--'x|
~ /-z~,OR1
~.~-CHzCHzOC ~-(x~ORz CHzCH ~
'OR 3
0.08 (1) 6. (2) >10. (3)
* Data from Ref. 25; number of H-bonding groups in parentheses.
J 5{
|
|
Fig. 11. The fit of bisquatemary compounds to the postulated choline binding site and the extent of inhibition of choline uptake.
probability of the occurrence of the "catastrophic" (30) all-or-none response of the entire apparatus that is re-
quired for transport to occur. Reassociation of transporter and controllers could occur as soon as the transporter
526
Roberts and Tamaru -8
A |
|
(CHsCH2)3 N(CH2)nN(CH2CHs)5 0
~-7 Q)
.~
-6
(CHs)z,N(CH2)nN{CH3)3
(CH3)31GNCnHzn+1
-5 ,x
X
0 X
s s
-..4 -4
-3
I
I
I
2
4
6
~
I
I
I
L
I
I
8 10 12 14 16 18 20 n
Fig. 12. Effects of chain length on inhibition of choline uptake by alkyl bis-trimethyl and bis-triethyl ammonium salts (from Ref. 26) and by alkyl trimethylammonium and dimethylammonium salts (from Ref.
9).
returns to its aboriginal state, beginning another cycle of activity. The rate-limiting step in transport is presumed to be the attachment of choline to an ~x site (Figure 13A), for which it has a high affinity (Km = 1.15 tzM (9)). A remarkable common feature of the bilobulate periplasmic binding proteins of bacterial transport systems is that their substrate specificities are conferred primarily by H-bonding interactions either with amino acid sidechains or with main-chain peptide NH and CO groups of the transporter protein (10). Although of far lesser precision than the above high-resolution crystallographic analyses, our observations are compatible in that they show that H-bonding also plays a key role in the binding of choline to the synaptosomal choline transporter (Figure 7 and Tables II and III). The model predicts that each one of the two identical or closely similar anionic o~ sites in the active un-
bound state of the transporter (Figure 13A) has approximately the same probability of combining with externally available ligand, but that in a particular transport cycle it has only one chance in two of being the site with which initial association of ligand takes place. Subsequently to attachment to one site, both sites cooperate in the rapid and irreversible transport of one molecule of ligand into the interior of a membrane-bounded volume (Figure 13B and 13C). Although there are no data with regard to the mechanisms of the latter, we suggest as one possibility that the closed-liganded form (Figure 13B) undergoes a configurational change that opens the Venus fly trap (Fig. 13C), previously closed in a high Na + and C1- and low K + extracellular environment, to the low Na + and CI- and high K § intracellular environment by temporary rupture of the hydrophobic association between the extensions of the a sites proposed above. The initial steps of choline binding would take place in an extracellular phase with the following ionic distribution (raM): Na + 150, K § 5.5, C1- 125 (Figure 13A). When configurationaI changes take place during choline uptake (Fig. 13C), the choline binding region of the transporter becomes exposed to the intracellular phase with the composition (raM): Na § 15; K § 150; C1- 9. The bound choline would immediately dissociate from the transporter and diffuse into the intracellular fluid, complete dissociation being insured by the combined effects of high choline-displacing K § concentrations and the low levels of Na § and CI-, which even in the absence of K + ions would be inadequate to sustain choline binding to the transporter. Metabolic maintenance of the above ionic asymmetries and, therefore, of the membrane potential across the membrane is achieved by operation of ionic pumps in coordination with appropriate sequences of opening and closing of related ion channels. The ionic gradients and resultant membrane potential, in turn, maintain the transporter in a choline-receptive configuration, furnishing the appropriate ionic environments necessary for the binding of choline to take place on the outside of the membrane and for its intracellular release. Particularly because of its high intracellular concentration, K § is an effective ligand for the choline transporter and is countertransported with choline (7,31). The countertransport, we suggest, takes place by reversal of the processes by which choline is transported inward. K § is presumed to exchange with choline during its release (Fig. 13C) and to undergo processes leading to its extracellular extrusion, as indicated in Fig. 13. Because there are such high intracellular concentrations of K § and low levels of free
Modeling the Ligand Binding Site of Synaptosomal Choline Transporter
527
OUTSIDE High No+ ond CI-, Low K + A.
K+
Chel:~
B.. ~ ; ! ~ , , ~ ~
g~f#
C. ;~r
~,}
D. Inactivafior~
ChoI.+
'~tivoflnq_ FoctQrs
Low No+ ond el-, High K "
K+ Chol+
Q Prolonation Depolorization
Phosphorylotion AIIosteric Ef fecfors
INSIDE
ATP Others
Fig. 13. Model for choline-K* countertransport. The choline and K § pathways are designated by the labels over the arrows. A, B, and C may undergo inactivation-reactivation. However, for the sake of simplicity, only a relationship of C with an inactivated form, D, is indicated on the diagram.
choline and because Na+-K+-ATPase is highly active, it would be extremely difficult to detect changes in concentrations or fluxes of K + during choline uptake. Rather, the roles of K + have been ascertained by studying the effects of K + on choline uptake and flux in various experimental paradigms. Factors suggested in the literature to possibly be involved in active-inactive transformations of the choline transporter (Fig. 13C and 13D) are protonation (11), depolarization (6,32,33), phosphorylation (3), and allosteric effects (34). It is beyond the scope of this paper to discuss these in detail. The uptake process as outlined in Figure 13 is similar in principle to ingestion of nutrient by an organism, entry of sperm into an ovum, or phagocytosis of a particle by a macrophage. For example, the unliganded transporter may be viewed as an open mouth (Fig. 13A) that closes on food or drink (Fig. 13B) which then is swallowed (Fig. 13C). In this manner macro mimics micro.
Recent progress in cloning bacterial (35) and yeast (36) choline transport genes indicates that some of the suggestions made in this paper soon will be testable with the elegant tools of molecular biology and that pure choline transporter protein will become available for detailed structural analysis.
ACKNOWLEDGMENTS
M. T. was the recipient of a fellowship from the California Foundation for Biochemical Research. We are most grateful to Ms. Markie Ramirez for careful preparation of the photographs in this paper.
REFERENCES 1. Simon, J. R., Atweh, S., and Kuhar, M. J. 1976. Sodium-dependent high affinity choline uptake: a regulatory step in the synthesis of acetylcholine. J. Neurochem. 26:909-922. 2. Jope, R. S. 1979. High affinity choline transport and acetylcoA production in brain and their roles in the regulation of acetylcholine synthesis. Brain Res. Rev. 1:313-344. 3. Lijian, Y., Nishino, H., and Iwashima, A. 1988. Stimulation of choline transport in cultured cells induced by 12-O-tetradecanoylphorbol-13-acetate: one of the earliest phenomena induced by the tumor promoter. Oncology 45:326-330. 4. Breer, H. 1983. Choline transport by synaptosomal membrane vesicles isolated from insect nervous tissue. FEBS Letters 153:345348. 5. Meyer, E. M., and Cooper, J. R. 1982. High-affinity choline transport in proteoliposomes derived from rat cortical synaptosomes. Science 217:843-845. 6. O'Regan, S., and Vyas, S. 1986. Modifications in choline transport activity as a function of membrane potential and the sodium gradient. J. Physiol., Paris 81:325-331. 7. Vyas, S., and O'Regan, S. 1985. Reconstitution of carrier-mediated choline transport in proteoliposomes prepared from presynaptic membranes of Torpedo electric organ, and its internal and external ionic requirements. J. Membrane Biol. 85:111-119. 8. Meyer, E. M., West, C. M., and Chau, V. 1986. Antibodies directed against ubiquitin inhibit high affinity [3H]choline uptake in rat cerebral cortical synaptosomes. J. Biol. Chem. 261:1436514368. 9. Tamaru, M., and Roberts, E. 1988. I. Structure-activity studies on inhibition of choline uptake by a mouse brain synaptosomal preparation: basic data. Brain Res. 473:205-226. 10. Quiocho, F. A. 1990. Atomic structures of periplasmic binding proteins and the high-affinity active transport systems in bacteria. Phil Trans. R. Soc. Lond. B 326:341-35t. 11. Deves, R., Reyes, G., and Krupka, R. M. 1986. The carrier reorientation step in erythrocyte choline transport: pH effects and the involvement of a carrier ionizing group. J. Membrane Biol. 93:165-175. 12. Krupka, R. M., and Deves, R. 1988. The choline carrier of erythrocytes: location of the NEM-reactive thiol group in the inner gated channel. J. Membrane Biol. t01:43-47. 13. Deves, R., and Krupka, R. M. 1987. Effects on transport of rapidly penetrating, competing substrates: activation and inhibition of the choline carrier in erythrocytes by imidazole. J. Membrane Biol. 99:13-23.
528 14. Krupka, R. M. 1989. Testing transport models and transport data by means of kinetic rejection criteria. Biochem. J. 260:885--891. 15. Mao, B., Pear, M. R., McCammon, J. A., and Quiocho, F. A. 1982. Hinge-bendingin L-arabinose-bindingprotein. J. Biol. Chem. 257:1131-1133. 16. Vos, J., Kuriyama, K., and Roberts, E. 1968. Electrophoretic mobilities of brain subcellular particles and binding of -,/-aminobutyric acid, acetylcholine, norepinephrine, and 5-hydroxytryptamine. Brain Res. 9:224-230. t7. Kuriyama, K., Roberts, E., and Vos, J. 1968. Some characteristics of binding of ",/-aminobutyricacid and acetylcholine to a synaptic vesicle fraction from mouse brain. Brain Res. 9:231252. 18. Vos, J., Kuriyama, K., and Roberts, E. 1969. Distribution of acid mucopolysaccharides in subcellular fractions of mouse brain. Brain Res. 12:172-179. 19. Krupka, R. M., and Deves, R. 1980. The electrostatic contribution to binding in the choline transport system of erythrocytes. J. Biol. Chem. 255:8546-8549. 20. Deves, R., and Krupka, R. M. 1979. The binding and translocation steps in transport as related to substrate structure. A study of the choline carrier of erythrocytes. Biochim. Biophys. Acta 557:469-485. 21. Martin, K. 1969. Effects of quaternary ammonium compounds on choline transport in red cells. Br. J. Pharmacol. 36:458--469. 22. Irwin, M. J., Nyborg, J., Reid, B. R., and Blow, D. M. 1976. The crystal structure of tyrosyl-transfer RNA synthetase at 2.7/~ resolution. J. Mol. Biol. 105:57%586. 23. Fersht, A. R., Mulvey, R. S., and Koch, G. L. E. 1975. Ligand binding and enzymic catalysis coupled through subunits in tyrosyltRNA synthetase. Biochemistry 14:13-18. 24. Bosshard, H. R., Koch, G. L. E., and Hartley, B. S. 1975. Aminoacyl-tRNA synthetases from Bacillus stearothermophilus. Asymmetry of substrate binding to tyrosyl-tRNA synthetase. Eur. J. Biochem. 53:493-498. 25. Lindborg, B., Crona, K., and Dahlbom, R. 1984. Troxoniumlike inhibitors of the high affinity uptake of choline in mouse brain synaptosomes in vitro. Acta Pharm. Suec. 21:27t--294.
Roberts and Tamaru 26. Holden, J. T., Rossier, J., Beaujouan, J. C., Guyenet, P., and Glowinski, J. 1975. Inhibition of high-affinity choline transport in rat striatal synaptosomes by alkyl bisquaternary ammonium compounds. Molecular Pharmacology 11:19-27. 27. Barker, L. A., and Mittag, T. W. 1975. Comparative studies of substrates and inhibitors of choline transport and choline acetyltransferase. J. Pharm. Exptl. Therap. 192:86-94. 28. Liron, Z., Wong, E., and Roberts, E. 1988. Studies on uptake of 3,-amiuobutyric acid by mouse brain particles; toward the development of a model. Brain Res. 444:119-132. 29. Zilberstein, D., Ophir, I. J., Padan, E., and Schuldiner, S. 1982. Na § gradient-coupled porters of Escherichia eoli share a common subunit. J. Biol. Chem. 257:3692-3696. 30. Thorn, R. 1975. Structural Stability and Morphogenesis (trarisluted from the French edition, as updated by the author, by D. H. Fowler; Foreword by C. H. Waddington), W. A. Benjamin, Reading, MA. 31. Martin, K. 1972. Extracellular cations and the movement of choline across the erythrocyte membrane. J. Physiol. 224:207-230. 32. Saltarelli, M. D., Lowenstein, P. R., and Coyle, J. T. 1987. Rapid in vitro modulation of [3H]hemicholinium-3 binding sites in rat striatal slices. Eur. J. Pharmacol. 135:35--40. 33. Antonelli, T., Beani, L., Bianchi, C., Pedata, F., and Pepeu, G. 1981. Changes in synaptosomal high affinity choline uptake following electrical stimulation of guinea-pig cortical slices: effect of atropine and physostigmine. Br. J. Pharmaeol. 74:525-531. 34. Hatch, G. M., Stevens, W. K., and Choy, P. C. 1988. Effect of amino acids on choline uptake and phosphatidylcholine biosyntehsis in the isolated hamster heart. Biochem. Cell Biol. 66:418424. 35. Andresen, P. A., Kaasen, I., Styrvold, O. B., Boulnois, G., and Strom, A. R. 1988. Molecular cloning, physical mapping and expression of the bet genes governing the osmoregulatory cholineglycine betaine pathway of Escherichia coli. J. Gen. Microbiol. 134:1737-1746. 36. Nikawa, J.-I, Hosaka, K., Tsukagoshi, Y., and Yamashita, S. 1990. Primary structure of the yeast choline transport gene and regulation of its expression. J. Biol. Chem. 265:15996-16003.