The nicotinic cholinergic receptor: a theoretical model.

Based on published affinity-labeling and mutagenesis experiments describing the effect of changes in specific amino acids in molecular biological studies on the nicotinic acetylcholinergic receptor (nAChR), we have identified 12 amino acids which are important in functioning at the nicotinic cholinergic receptor. The work presented here provides an atomistic model of this important receptor based on our molecular modeling studies. We found five of these amino acids (TRP86, ASP89, TYR93, ASP138, and THR191) to be associated with the cationic end of acetylcholine (ACh), which is electron-deficient. Three other amino acids (ARG209, TYR190, and TYR198) are associated with the ester end, where an enhanced electron density is present. After hydrogen bonding between the two oxygen atoms at the ester end, and two of the guanidinium hydrogen atoms in ARG209. ASP200 hydrogen bonds to the other two hydrogen atoms of the guanidinium group, thus forming a pseudo-ring. Two aromatic amino acids (TRP149 and TYR151) then enhance the binding at the pseudo-ring through additional hydrogen bonding and charge-transfer complexation, with THR150 functioning to further stabilize this evolving charge-transfer complex. We postulate that this latter process allows the ion channel to twist, thus opening it. From the published amino acid sequence in the polypeptides at the 5HT-3, GABA, and glycine receptors (Maricq et al.: Science 254:432-437, 1991), we also speculate on which amino acids are involved in these three receptors.

Glycine and GABA receptors: molecular mechanisms controlling chloride ion flux.

We have been able to show that the three clearly identified atoms common to the inhibitory neurotransmitters glycine and GABA, that we previously hypothesized to serve as attachment points at the glycinergic and gabanergic receptor, can indeed interact through both electrostatic and hydrogen bonding to several amino acids, which have been identified in molecular biological investigations as both present and critical in the physiological functioning of key polypeptides common to these inhibitory receptors. In addition, amino acids also involved in stabilizing the interaction between the antagonists strychnine and R5135 at the glycinergic and gabanergic receptors, respectively, have been shown to fit our complex model. We identify in detail molecular mechanisms to explain how glycine and GABA initiate chloride ion movement from extraneuronal fluid in the synaptic cleft to intraneuronal volume. In addition, we also identify the molecular mechanisms involved in the blocking of chloride ion movement by strychnine at the glycinergic receptor and by R5135 at the gabanergic receptor. We also present two computer-generated color prints, one for the glycine receptor and one for the GABA receptor, which show the quantum mechanically geometry optimized complex formed between receptor side chains, i.e., the part of the amino acids in the polypeptide that interacts with the zwitterionic inhibitory neurotransmitters. These computer-generated color figures also show a) the important electrostatic and hydrogen bonding in these interactions, b) a van der Waals model of this complex to illustrate that no steric repulsions exist, and c) the molecular electrostatic potential energy map showing the electrostatic potentials of neurotransmitter bound to the receptor model. Finally, we show with computer calculations that the pseudo-rings, formed between the positive quanidinium group in arginine and one of the oxygen atoms in the carboxyl group in both glycine or GABA, result in a positive planar region which appears to be involved in a charge-transfer complex with aromatic benzene groups in amino acids such as phenylalanine and tryosine.

Comparison of binding mechanisms at cholinergic, serotonergic, glycinergic and GABAergic receptors.

Employing computational methods and published data from molecular biological studies involving amino acid sequences in the polypeptide receptors, the authors studied and compared how two excitatory neurotransmitters, ACh and 5-HT, and two inhibitory neurotransmitters, glycine and GABA, can bind to their respective recognition sites at CNS receptors. Models for each neurotransmitter interaction with specific amino acids are described and identified. Molecular mechanisms are identified that can explain how the binding process initiates ion flow through channels located within the postsynaptic membrane such that if the neurotransmitter is inhibitory, hyperpolarization occurs, and if excitatory, depolarization occurs. Although the theoretical work described indicates that there is a difference in molecular mechanisms operative at the anionic and cationic channels, and provides an explanation why the former is more specific, the molecular modeling data and the similarities of specific amino acids in the sequence in all four receptor polypeptides used to construct the four models support ACh, 5-HT, glycine and GABA as being members of the same ligand-gated ion channel superfamily.

Identifying agonistic and antagonistic mechanisms operative at the GABA receptor.

Based on our molecular modeling investigations of the glycinergic receptor, we expanded our studies to similarly investigate the GABAergic receptor. New data suggest there may exist a slightly different agonistic mechanism for the molecules described herein as compared to glycine. The origin of this is undoubtedly the fact that, while glycine has a positive and two negative binding sites, it is significantly shorter than GABA and the other GABA agonists. Clearly, discovery of more glycine agonists is needed to further clarify this point. Moreover, we find a remarkedly different antagonistic mechanism exists for this phylogenetically newer inhibitory system in the central nervous system (CNS) than recently reported for strychnine and eight weaker glycine antagonists. We used GABA and six agonists (muscimol, dihydromuscimol, THIP, isoguvacine, trans-3-aminocyclopentane-1-carboxylic acid, piperidine-4-sulfonic acid) and five antagonists (bicuculline-N15-methobromide, R5135, pitrazepin, iso-THAZ and securinine) to derive our conclusions. We found that each of the agonists have three clearly defined atoms that can serve as attachment points at the GABAA receptor site. One of the three attachment atoms includes a carbonyl or carboxylate oxygen. The role of the carbonyl or carboxylate atom is very important. First, we theorize that a rapid two-point attachment occurs (one from the positive end and one from one of the other two negative atoms on the ligand) at the recognition site in the receptor where GABA or a GABAergic agonist binds. The positive end of the agonist perhaps associates through hydrogen bonding to a beta-carboxyl group in one of the aspartate molecules in the polypeptide. The negative attachment points perhaps bind through hydrogen bonding to arginine molecules in this polypeptide. The second negative site in the agonist immediately triggers a conformational change by pulling together the aforementioned groups by electrostatic attraction, and hence opening the chloride channel. We propose the carbonyl oxygen is partly responsible for triggering the opening by formation of a double hydrogen bond to arginine. We postulate that this attraction is the first step inducing the conformational change. In the case of the GABA antagonists investigated, a fourth attachment site was not found. In fact only two sites have been identified similar to the group II glycine antagonists. Our data support a hypothesis for GABAergic antagonist activity which suggests that the antagonist simply binds to the recognition site and blocks the neurotransmitter, GABA, from entering this site thereby preventing the opening of the chloride channel; it just stays closed. This mechanism is different from the mechanism proposed for the large number of Group I glycine antagonists (Aprison et al.: J Neurosci Res 41: 259-269, 1995).

Operant response suppression induced with systemic administration of 5-hydroxytryptophan is centrally mediated.

Intracerebroventricular (ICV) administration of selective serotonergic agents was used to examine the extent of central mediation of 5-HTP-induced operant response suppression in rats. ICV administration of LY53857 (1.0, 3.75, or 7.5 micrograms/5 microliters/5 min) dose dependently blocked response suppression induced with systemically administered 5-HTP (25 mg/kg, IP), whereas ICV 0.9% saline (5 microliters over 5 min) had no significant effect on 5-HTP-induced response suppression. ICV ketanserin (7.5 micrograms/5 microliters/5 min) also blocked response suppression induced with systemically administered 5-HTP. ICV administration of the 5-HT2A/2C receptor agonist DOI (80 micrograms/5 microliters/5 min) induced significant periods of response suppression in this model, which was blocked with LY53857 (1.0 mg/kg, IP) pretreatment. These data demonstrate that central administration of 5-HT2A/2C antagonists potently attenuate operant response suppression induced with systemically administered 5-HTP or DOI and are in agreement with previous findings suggesting central mediation of 5-HTP-induced operant response suppression.

On identifying a second molecular antagonistic mechanism operative at the glycine receptor.

We used molecular modeling techniques to examine six reported antagonists of glycine with varying Ki values against strychnine. We found the data suggest two groups operating with different mechanisms. In group 1 (strychnine, brucine, Pitrazepin, and bicuculline methobromide) the antagonist contains two or three sites that can electrostatically bind to the three comparable groups of opposite charge in the recognition site where the natural neurotransmitter binds, thus opening the chloride channel. In addition, when in this position, the antagonist is able to also block the now opened chloride channel with a different portion of its structure. In many cases, this involves an interaction between a carbonyl group on the antagonist and the guanidinium group of arginine which is part of the polypeptide segment of the outer mouth of the chloride channel (Grenningloh et al., Nature 330:25-26, 1987). In group 2 (R5135 and 1,5-diphenyl-3,7-diazaadamantan-9-ol) the antagonist contains charged sites but when one of these molecules attaches to the recognition site, the chloride channel is not opened. In addition, R5135 contains a carbonyl group which attaches to arginine as pointed out in the text, whereas 1,5-diphenyl-3,7-diazaadamantan-9-ol contains a phenyl group that can block the channel.

On a molecular comparison of strong and weak antagonists at the glycinergic receptor.

Using molecular modeling techniques, we studied nine glycine antagonists in order to try to identify the molecular descriptors that characterize strychnine as a strong antagonist and N,N-dimethyl-muscimol, iso-THIA, THIA, N-methyl-THIP, iso-THAZ, THAZ, iso-THPO, and iso-THAO (see Experimental for chemical names) as weak glycine antagonists. We confirm that all nine compounds have the three-atom regions (two negative and one positive) that we have postulated are necessary to permit such compounds to attach to the recognition site in the glycinergic synapse. Furthermore, in the case of antagonists we have postulated the presence of a fourth atom that can attach to the top of the chloride ion channel. Each of the nine antagonists has such a fourth negative atom and the latter property gives each of these compounds their antagonistic characteristic. Further, only in the case of strychnine is there evidence that at its positively charged end does the positive charge extend to cover a region that could bind through electrostatic domains to a tertiary carboxyl group in an amino acid like aspartate. Published molecular biological data show that such an amino acid is present in the portion of the polypeptides identified in the glycine receptor. The bidentate binding is superior to the single site attachment that is present in the other eight weak glycine antagonists. In addition, the two negative atom sites in each antagonist are also in a position to participate in electrostatic binding through bidentate involvement with the positively charged guanidinium group of arginine. The latter amino acid also has been identified in the portion of the polypeptide chain at the glycine receptor. Finally, our molecular data predict that after strychnine, the eight weak glycine antagonists listed above are in order of decreasing potency, i.e., N,N-dimethyl-muscimol is the best of the weak antagonists and iso-THAO should be the weakest.

Identification of a second glycine-like fragment on the strychnine molecule.

Strychnine is a complex molecule that inhibits the physiological actions of glycine, an important inhibitory neurotransmitter in the spinal cord, brain stem, and other areas of many vertebrates. Since 1987, we have employed atomistic molecular modeling tools to find an explanation at the molecular level for how this antagonism works. We have located a second glycine-like fragment in the strychnine molecule that, when compared to glycine in a three pair atom analysis, provides an excellent topological and electronic charge congruence. The topological congruence in the second glycine-like fragment is much better than with the first fragment reported in 1987 when using a truncated strychnine molecule in the quantum mechanical analysis. A fourth negative atom, a characteristic of antagonists which we reported earlier (Aprison and Lipkowitz: J Neurosci Res 30:442-446, 1991; Aprison and Lipkowitz: J Neurosci Res 31:166-174, 1992) was found in strychnine. This result follows the pattern reported recently for the three weak glycine antagonists N,N-dimethylmuscimol, N-methyl-THIP, and iso-THAO, a bicyclic 5-isoxazolol zwitterion.

Muscimol and N,N-dimethylmuscimol: from a GABA agonist to a glycine antagonist.

By using molecular modeling methods, a molecular mechanism was identified which can explain how the incorporation of two methyl groups in place of two hydrogen atoms on the terminal nitrogen atom of muscimol can not only convert this potent agonist at GABAnergic receptors to an inactive molecule at these receptors, but also can convert this new derivative to an antagonist of glycine at glycinergic receptors. This insight into the molecular mechanism operative in the conversion of physiological function provides a basis for understanding how a single molecule may be able to act at both the GABA- and glycine-inhibitory receptors.

Molecular modeling of the weak glycine antagonist iso-THAO.

When compared to strychnine, a potent glycine antagonist, iso-THAO, a bicyclic 5-isoxazolol zwitterion, has been reported to be a weak glycine antagonist. Since there are so few glycine antagonists, and there is a striking lack of similarity between the structures of these two antagonists, iso-THAO was studied using current molecular modeling techniques and quantum mechanical calculations in order to compare the structural features and charge distributions of iso-THAO with glycine. The results of this study confirm our earlier hypothesis that an antagonist to inhibitory neurotransmitters like glycine and GABA has at least three binding sites to the natural receptor that are very similar to three such binding sites in the transmitter and its agonists, and each antagonist has an additional negative binding site. We speculate that the latter negative binding site can attach to the top of the chloride channel within the receptor complex. The diminished inhibitory activity of iso-THAO is attributed to its poor structural congruence with the three atom attachment sites used by glycine at its natural recognition site.

Differential effect of 5-hydroxytryptophan on approach and avoidance behavior in rats.

The current hypersensitive postsynaptic serotonin receptor theory of depression developed and expanded by Aprison and Hingtgen was based on an animal model of behavior in which food-reinforced approach behavior was suppressed following 5-hydroxytryptophan (5-HTP) administration. In this paper, data are presented to show that when the same animal is taught to emit, alternatingly, approach and avoidance behavior, and the serotonin precursor, 5-HTP, is administered, only the approach behavior is affected. Adult, male Wistar rats were trained on Sidman avoidance (RS20:SS10) and food-reinforced approach (VI 1) schedules. During the first part of this study, rats received separately 50-min sessions for approach and avoidance responding. For the second part, both schedules were given in the same experimental chamber. In the third part, 10-min alternating approach and avoidance components were combined in the same 50-min sessions. Significant behavioral suppression of approach responding was observed following administration of L-5-HTP (50 mg/kg IP), as well as after D,L-5-HTP (25 and 50 mg/kg IP) in a dose-dependent relationship, whereas no significant effect was seen for Sidman avoidance responding during this type of session. These results support the role of serotonin in food-reinforced approach behavior and suggest that suppression of Sidman avoidance behavior may be mediated by other neurotransmitter systems.

Tetrahydroaminoacridine (THA) as a pharmacological probe in Alzheimer's disease (AD) and other neurodegenerative disorders.

Unlike other potent enhancers of cholinergic function in the central nervous system (CNS), THA appears to sustain improved function in many moderately impaired AD patients when the Summers procedure is followed. THA has a complex pharmacology. In addition to its enhancement of cholinergic transmission a hydroxylated metabolite might chelate aluminum (A1), thereby removing multiple toxicological constraints on CNS function. This mobilized THA metabolite-A1 complex might either be re-distributed to less sensitive sites or removed from the CNS across the blood-brain barrier (BBB). Since the known presence of A1 in AD brain is not necessarily causal, a positivistic approach to research and treatment with THA and its metabolites might serve to clarify this difficult and challenging problem.

Activity-wheel stress and serotonergic hypersensitivity in rats.

Adult male Wistar rats were subjected to activity wheel stress: unlimited access to an activity wheel for up to twelve days and food for 30 to 60 min each day. Each treated rat was paired with a control, the latter being housed in home cages and given sufficient food to maintain a weight similar to the stressed partner. All rats were previously trained on a variable interval schedule for milk reinforcement. When the activity of the stressed rat increased rapidly then decreased suddenly, the pair was decapitated for biochemical analysis. Levels of the serotonin metabolite, 5-hydroxyindoleacetic acid, decreased by 50%, and the Bmax for ketanserin binding increased by 19% in frontal cortical homogenates from the stressed rats when compared to controls. These data support the concept that stress increases the sensitivity of central serotonin receptors.

On the GABAA receptor: a molecular modeling approach.

Bicuculline methobromide, a complex alkaloid, antagonizes in some unknown manner the action of GABA, an important inhibitory transmitter in the CNS. To help understand the mechanism of this antagonism we have employed molecular modeling techniques to assess the similarity and difference between this antagonist and GABA plus four direct GABA agonists. Topological and electronic charge congruence between these five molecules was examined in great detail. It was found that each of the five molecules has three clearly defined atoms that serve as attachment points at the GABAA receptor site. It is hypothesized that an additional negatively charged atom on bicuculline serves as an additional point of attachment that blocks the nearby chloride ion channel. The model presented offers an explanation of why muscimol acts as a better agonist than GABA as well as rationalizing why (+)-bicuculline acts as an antagonist but (-)-bicuculline does not.

Response suppression in rats after bilateral microinjection of 5-hydroxytryptophan in lateral hypothalamus.

Studies using the 5-hydroxytryptophan (5-HTP) animal model of depression have led to the development of the hypersensitive postsynaptic serotonin receptor theory of depression. To demonstrate more clearly that the 5-HTP-induced suppression is a centrally mediated phenomenon, rats were implanted with bilateral cannulae in the lateral hypothalamus and received microinjections of D,L-5-HTP (100-500 ng) 15 min after the start of a VI operant session (milk reinforcement). Significant decreases in responding were observed that were comparable to those obtained after a systemic injection of 50 mg/kg D,L-5-HTP. Rats receiving a microinjection of 5-HTP in the posterior hypothalamus did not exhibit a behavioral effect. Rats working on shock-avoidance schedules did not demonstrate response suppression following microinjection of 5-HTP into the lateral hypothalamus, which is the same result as that following systemic 5-HTP administration. These data support the important role previously assigned to central 5-HT mechanisms in the 5-HTP animal model of depression.

Potentiated 5-hydroxytryptophan induced response suppression in rats following chronic reserpine.

Since reserpine precipitates depression in some hypertensive patients, we tested this drug on our animal model of depression. The present experiment was designed to measure the effects of chronic reserpine treatment on 5-hydroxytryptophan (5-HTP) induced behavioral depression in rats trained on a food reinforcement operant schedule. Based on the Aprison et al. model of depression involving the serotonergic system, we predicted the development of receptor supersensitivity of postsynaptic serotonin (5-HT) receptors due to the decreased release of this neurotransmitter as a consequence of chronic reserpine treatment. Rats were trained on a VI 1 reinforcement schedule and then divided into 3 chronic treatment groups. One received daily injections of a placebo, another 0.025 mg/kg reserpine and the third 0.05 mg/kg reserpine. We found that 5-HTP induced behavioral depression was potentiated in rats chronically treated with reserpine, thus suggesting the development of supersensitive 5-HT receptors. These results support the hypothesis that in some types of human depression a decreased release of 5-HT occurs of sufficient duration to permit the subsequent development of supersensitive 5-HT receptors.

Identification of a glycine-like fragment on the strychnine molecule.

Strychnine, a complex molecule, antagonizes in some unknown manner the action of glycine, an important inhibitory neurotransmitter in the spinal cord and brainstem of many vertebrates. To help understand the mechanism of this antagonism, we have employed modern computational methods to assess the similarities between these seemingly different molecules. An exhaustive comparison of topological and electronic features of both molecules was made. We have successfully located a glycine-like fragment in the strychnine molecule that, when compared to glycine, exhibits both topological and electronic charge congruence. The successful location of this glycine-like fragment allows us to speculate how the large strychnine molecule assumes its role as an antagonist against the inhibitory action of glycine, the simplest amino acid.

Choline acetyltransferase and glutamate uptake in the nucleus tractus solitarius and dorsal motor nucleus of the vagus: effect of nodose ganglionectomy.

Unilateral removal of the nodose ganglion resulted in a significant decrease in choline acetyltransferase activity in the ipsilateral dorsal motor nucleus of the vagus but was without effect on enzyme activity in the nucleus of the solitary tract. High affinity glutamate uptake in the dorsal motor nucleus of the vagus and along the rostrocaudal extent of the nucleus of the solitary tract was not affected by nodose ganglionectomy.

Blockade of a 5-hydroxytryptophan-induced animal model of depression with a potent and selective 5-HT2 receptor antagonist (LY53857).

To test the hypothesis that a new potent and selective 5-HT2 receptor antagonist would be an excellent blocker of D,L-5-hydroxytryptophan (5-HTP)-induced response suppression in an animal model of depression, we administered LY53857 60 min prior to 5-HTP injections into rats working on an operant schedule for milk reinforcement. As predicted, LY53857 pretreatment significantly blocked 5-HTP depression (90%) in doses as low as 0.1 mg/kg ip. When the dose of LY58357 was further reduced to 0.025 mg/kg, blockade of 5-HTP-induced depression was still greater than 30%. In doses as high as 5.0 mg/kg, LY53857 alone had no effect on the baseline performance of rats working a VI 1 schedule. Pretreatment with desipramine (2.5 mg/kg), an antidepressant characterized as having major noradrenergic effects, did not significantly block the 5-HTP-induced depression. These data suggest that the 5-HTP-induced depression is mediated by serotonergic mechanisms involving 5-HT2 receptors, as LY53857 is a selective antagonist of these receptors. These data also support the suggestion, based on other published data from this laboratory, that some antidepressants are antagonizing 5-HT2 receptors in our animal model of depression and may also act in a similar manner in depressed patients. Thus, this new drug could be of interest as a possible antidepressant agent of the general type that was proposed earlier by Aprison and Hingtgen (1981).