Inhibition of gastric (H+ + K+)-ATPase by unsaturated long-chain fatty acids.

Arachidonic acid and unsaturated C18 fatty acids at concentrations near 10(-5) M markedly inhibited (H+ + K+)-ATPase in hog or rat gastric membranes. Arachidonic acid was a more potent inhibitor than unsaturated C18 fatty acids, but the involvement of the metabolites of arachidonic acid cascade was ruled out. Linolenic acid inhibited the formation of phosphoenzyme and the K+ -dependent p-nitrophenylphosphatase activity of the hog ATPase. Treatment with fatty acid-free bovine serum albumin abolished only the inhibitory effect of the fatty acid on the phosphatase activity without restoring the overall ATPase action. These data suggest the existence of at least two groups of hydrophobic binding sites in the gastric ATPase for unsaturated long-chain fatty acids which affect differentially the catalytic reactions of the ATPase. (H+ + K+)-ATPase in rat gastric membranes was found more susceptible to the fatty acid inhibition and also more unstable than the ATPase in hog gastric membranes. The presence of a millimolar level of lanthanum chloride or ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid stabilized the rat ATPase probably via the inhibition of Ca2+ -dependent phospholipases in the gastric membranes.

Heterogeneity of histidine transport in the Ehrlich cell.

We have reexamined the heterogeneity shown by histidine in its uptake by the Ehrlich ascites tumor cell, in the face of a contradiction of our earlier interpretation. We again find the fraction of histidine uptake at neutral pH inhibitable by the model substrate for System A, 2-(methylamino)-isobutyric acid, to be fully dependent on the presence of Na+ or Li+. The small Na+ -independent component not attributable to System L can be identified with System Ly+ through its inhibitability by homoarginine. This component increases as the pH is lowered with an apparent pK' a of 6.1. The simultaneous decrease in the uptake by the neutral systems could be identified, for System L, with the same titration of histidine to its cationic form, but for System A the sharp decrease is identified with the protonation of a structure on the membrane rather than one on the substrate. The action of H+ in the latter case proved approximately non-competitive with Na+ when tested with ordinary substrates.

Reversal of antisecretory activity of omeprazole by sulfhydryl compounds in isolated rabbit gastric glands.

We have examined the interaction of omeprazole, a gastric antisecretory agent, with endogenous or exogenous sulfhydryl compounds in isolated rabbit gastric glands. The glands exposed to omeprazole (2 microM for 50 min) could recover acid secretory response to dibutyryl-cAMP upon addition of dithiothreitol, cysteine or glutathione. Washing the omeprazole-exposed glands free of the extracellular drug also led to a similar recovery of the acid secretory response. Depletion of cellular glutathione with 2-cyclohexen-1-one had no considerable effect on the secretory response of the glands to dibutyryl-cAMP, but prevented the reversal of the antisecretory effect of omeprazole upon washing or adding exogenous cysteine. Also, the antisecretory potency of omeprazole increased several fold in the glutathione-depleted glands. These observations indicate that cellular glutathione is essential to reactivate the omeprazole-modified enzyme(s), possibly (H+ + K+)-ATPase, in acid secretory process and led us to propose that omeprazole is an agent reacting with sulfhydryl groups.

Amino acid stimulation of ATP cleavage by two Ehrlich cell membrane preparations in the presence of ouabain.

Two membrane fractions prepared from the Ehrlich ascites-tumor cell show non-identical stimulatory responses to certain amino acids in their Mg+2 -dependent activity to cleave ATP, despite the presence of ouabain and the absence of Na+ or K+. The first of these, previously described, shows little (Na+ + K+)-ATPase activity, and is characteristicallly stimulated by the presence of certain diamino acids with low pK2, and at pH values suggesting that the cationic forms of these amino acids are effective. The evidence indicates that these effects are not obtained through occupation of the kinetically discernible receptor site serving characteristically for the uphill transport of these amino acids into the Ehrlich cell. The second membrane preparation was purified with the goal of concentrating the (Na+ +K+)-ATPase activity. It also is stimulated by the model diamino acid, 4-amino-1-methylpiperidine-4-carboxylic acid, and several ordinary amino acids. The diamino acids were most effective at pH values where the neutral zwitterionic forms might be responsible. Among the optically active amino acids tested, the effects of ornithine and leucine were substantially stronger for the L than for the D isomers. The list of stimulatory amino acids again corresponds poorly to any single transport system, although the possibility was not excluded that stimulation might occur for both preparations by occupation of a membrane site which ordinarily is kinetically silent in the transport sequence. The high sensitivity to deoxycholate and to dicyclohexylcarbodiimide of the hydrolytic activity produced by the presence of L-ornithine and 4-amino-1-methyl-piperidine-4-carboxylic acid suggests that the stimulatory effect is not merely a general intensification of the background Mg+ -dependent hydrolytic activity.

Inhibition of (H+ + K+)-ATPase and H+ accumulation in hog gastric membranes by trifluoperazine, verapamil and 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate.

The mechanism of gastric antisecretory action for trifluoperazine, verapamil and 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) has been studied utilizing isolated hog gastric membranes enriched with (H+ + K+)-ATPase. The drugs inhibited the gastric ATPase due to their apparent competition with K+ for the luminal high-affinity K+-site of the ATPase. The dose to inhibit 50% (ID50) of the ATPase in the membranes rendered freely permeable to K+ (20 mM) was 50 microM for trifluoperazine and 1.5 mM for verapamil and TMB-8. In intact hog gastric membranes which develop a pH gradient in the presence of valinomycin, ATP and KCl, however, trifluoperazine at 4 microM, verapamil and TMB-8 at 15 microM inhibited 40 and 30% of the valinomycin-stimulated ATPase activity, respectively, and also blocked the ionophore-dependent intravesicular acidification as measured by aminopyrine accumulation. The enhanced potency of the drugs to inhibit the ATPase in the intact membrane vesicles may be attributed to the accumulation of the drugs as a weak base within the vesicles, where the luminal K+-site of the ATPase is accessible. Calmodulin and Ca2+ had no effect on the extent of H+-accumulation as measured by aminopyrine accumulation in the membrane vesicles which were prepared in the presence of 1 mM EGTA. Since the drugs showed similar potency in interfering with H+ movements either in the membrane vesicles or isolated rabbit gastric glands stimulated by dibutyryl cAMP, it is reasonable to suggest the inhibitory effect of the drugs on (H+ + K+)-ATPase as a primary cause for such interferences in both cases. A trifluoperazine analog and other lipophilic amine drugs similarly inhibited (H+ + K+)-ATPase and H+ accumulation in the membrane vesicles or in the glands. We have concluded that a tertiary amine, the only common functional group among these drugs, is primarily responsible for their ability to interact with the high-affinity K+ site of the gastric ATPase.

Effect of carbachol or histamine stimulation on rat gastric membranes enriched in (H+-K+)-ATPase.

We have examined histamine- or carbachol-induced changes in rat gastric membranes enriched in K+-stimulated ATPase. Stimulation of secretion by both secretagogues in vivo produced a class of microsomal membranes which exhibited valinomycin-independent, KCl-dependent H+ transport. In contrast, membrane vesicles isolated from cimetidine inhibited resting mucosa exhibited largely the ionophore-dependent H+ transport. In addition, only in the carbachol-stimulated membranes a portion of the ionophore-independent H+ transport was refractory to cimetidine pretreatment. The gastric microsomal membranes were resolved into light and heavy fractions by centrifugation over isotonic 2H2O media. The ionophore-independent H+ transport was almost exclusively associated with the heavy microsomal fraction while the ionophore-dependent H+ transport was detected in the light fraction. Also, these fractions were considerably different from each other in their appearance in electron micrographs and SDS gel electrophoresis patterns. Secretagogue stimulation increased the population of the heavy microsomal membrane vesicles exhibiting the valinomycin-independent, K+-dependent H+ transport and their overall content of K+-stimulated ATPase. Cimetidine treatment, on the other hand, increased the ATPase activity associated with the light microsomes, and produced the heavy microsomal membranes showing only a marginal degree of the ionophore independent H+ accumulation, even though they were very similar to the carbachol-stimulated heavy membranes in the specific activity of K+-stimulated ATPase. SDS gel patterns and appearance in electron micrograph. These observations suggest that activation of secretion involves at least two distinctive events; transformation of the light to the heavy gastric membranes containing a K+-dependent H+ pump and an increased KCl permeability in the latter.

Gastric antisecretory activity of cycloheximide due to inhibition of protein synthesis.

Treatment of rats with cycloheximide 1 h before carbachol dose-dependently reduced the secretagogue-stimulated gastric acid secretion in pylorus ligated rats, and partially blocked carbachol- or histamine-induced activation of rat gastric (H+ + K+)-ATPase which includes translocation of reserve intracellular (H+ + K+)-ATPase into the apical membrane of the parietal cells and induction of a KCl pathway. Time-course studies showed that the drug was effective only when administered at least 30 min before the secretagogues. Puromycin showed the same effect as cycloheximide. Pulse labelling studies with [35S]methionine led to identification of two most actively synthesized polypeptides in rat gastric mucosa; the proteins of 38,000 and 14,000 molecular weight. The larger polypeptide was identified as rat pepsinogen. The identity of the smaller protein is not known yet. We suggest that synthesis of nascent polypeptide(s) is required for certain steps of the acid secretory process leading to the activation of the acid pump.

Aging: stimulation rate on cardiac intracellular Na+ activity and developed tension.

Previous reports suggested that Na,K-ATPase activity and Na(+)-pump capacity decrease with senescence in left atrial myocardium of F344 rats. Current experiments were designed to determine if this reduction in the Na(+)-pump affects free intracellular Na+ levels. Mean intracellular Na+ ion activity (aiNa) was measured with Na-selective microelectrodes in left atrial muscle isolated from hearts of 4-, 14- and 25-month-old F344 rats. Preparations were stimulated randomly at frequencies between 0 and 12 h. There were no age-associated differences in aiNa measured at any frequency or in the decay of Na+ activity following discontinuation of electrical stimulation. These data indicate that the aging-related decline in Na,K-ATPase does not result in elevated aiNa even at extremely high stimulation frequencies, thus suggesting that other routes of Na+ influx and efflux are also altered in atrial muscle.

Studies on (H(+)-K+)-ATPase inhibitors of gastric acid secretion. Prodrugs of 2-[(2-pyridinylmethyl)sulfinyl]benzimidazole proton-pump inhibitors.

The synthesis of N-substituted benzimidazole (H(+)-K+)-ATPase or proton-pump inhibitors is described. These compounds were prepared to function as prodrugs of the parent N-H compound and evaluated for their ability to inhibit gastric (H(+)-K+)-ATPase and gastric acid secretion. The prodrugs reported rely on either in vivo esterase hydrolysis for liberation of the parent compound (type I and type II) or require an acid environment for release of the active drug (type III and type IV). The N-(acyloxy)alkyl-substituted benzimidazoles 9, 11, and 24 showed improved chemical stability in the solid state and in aqueous solutions when compared to their parent N-H compounds. When given orally, 24 was found to be twice as potent as omeprazole in both the Shay rat and inactivation of gastric (H(+)-K+)-ATPase in the rat. The N-ethoxy-1-ethyl-substituted benzimidazoles 48-50 were found equally as effective as the N-H compound for inhibition of rat (H(+)-K+)-ATPase activity. In the Shay rat 48 at 10 mg/kg was approximately twice as active as parent timoprazole.

Antagonist, partial agonist, and full agonist imidazo[1,5-a]quinoxaline amides and carbamates acting through the GABAA/benzodiazepine receptor.

(4RS)-1-(5-Cyclopropyl-1,2,4-oxadiazol-3-yl)-12,12a-dihyd roimidazo[1,5- a]pyrrolo[2,1-c]quinoxalin-10(11H)-one (1a), 5-benzoyl-3-(5-cyclopropyl-1,2,4-oxadiazol-3-yl)-4,5- dihydroimidazo[1,5-a]quinoxaline (13b), and tert-butyl (4S)-12,12a-dihydroimidazo[1,5-a]pyrrolo[2,1- c]quinoxaline-1-carboxylate (1e), as well as other imidazo[1,5-a]quinoxaline amides and carbamates, represent a new series of compounds which bind with high affinity to the GABAA/benzodiazepine receptor. These compounds exhibit a wide range of intrinsic efficacies as measured by [35S]TBPS binding ratios. The synthesis of 1a begins with the addition of DL-glutamic acid to 1-fluoro-2-nitrobenzene, followed by reduction of the nitro group and subsequent ring closure to form 3-(carbethoxymethyl)-1,2,3,4-tetrahydroquinoxalin-2-one, followed by a second ring closure to afford (4RS)-1,5-dioxo-1,2,3,4,5,6-hexahydropyrrolo[1,2-a]quinoxali ne as the key intermediate. Appendage of a substituted imidazo ring via the anion of 5-cyclopropyl-1,2,4-oxadiazol-3-yl gives 1a. The (-)- and (+)-isomers of 1a were prepared from 1-fluoro-2-nitrobenzene and L- and D-glutamic acid, respectively. 1a and its enantiomers demonstrated affinity for the [3H]flunitrazepam binding site with Ki's of 0.87, 0.62, and 0.65 nM, respectively.

High-affinity alpha-aminobutyric acid A/benzodiazepine ligands: synthesis and structure-activity relationship studies of a new series of tetracyclic imidazoquinoxalines.

A series of tetracyclic imidazoquinoxaline analogs was developed which constrain the carbonyl group of the partial agonist 3-(5-cyclopropyl-1,2,4-oxadiazol-3-yl)-5-[(dimethylamino)carbonyl] - 4,5-dihydroimidazo[1,5-alpha]quinoxaline (2, U-91571) away from the benzene ring. These analogs orient the carbonyl group in the opposite direction of the previously reported full agonist 1-(5- cyclopropyl-1,2,4-oxadiazol-3-yl)-12,12a-dihydroimidazo[1,5- alpha]pyrrolo [2,1-c]quinoxalin-10(11H)-one (3, U-89267). A number of approaches were utilized to form the "bottom" ring of this tetracyclic ring system including intramolecular cyclizations promoted by Lewis acids or base, as well as metal-carbenoid conditions. The size and substitution pattern of the additional ring was widely varied. Analogs within this series had high affinity for the benzodiazepine receptor on the alpha-aminobutyric acid A chloride ion channel complex. From TBPS shift and Cl- current assays, the in vitro efficacy of compounds within this class ranged from antagonists to partial agonists with only 18a identified as a full agonist. Additionally, several analogs were quite potent at antagonizing metrazole-induced seizures indicating possible anticonvulsant or anxiolytic activity. Unlike 3, analogs in this series did not have high affinity for the diazepam insensitive alpha 6 beta 2 delta 2 subtype. These results suggest that either constraining the carbonyl group away from the benzene ring or the greater planarity that results from the additional cyclic structure provides analogs with partial agonist properties and prevents effective interaction with the alpha 6 beta 2 delta 2 subtype.

3-Phenyl-substituted imidazo[1,5-alpha]quinoxalin-4-ones and imidazo[1,5-alpha]quinoxaline ureas that have high affinity at the GABAA/benzodiazepine receptor complex.

A series of imidazo[1,5-alpha]quinoxalin-4-ones and imidazo[1,5-alpha]quinoxaline ureas containing substituted phenyl groups at the 3-position was developed. Compounds within the imidazo[1,5-alpha]quinoxaline urea series had high affinity for the GABAA/benzodiazepine receptor complex with varying in vitro efficacy, although most analogs were partial agonists as indicated by [35S]TBPS and Cl- current ratios. Interestingly, a subseries of piperazine ureas was identified which had biphasic efficacy, becoming more antagonistic with increasing concentration. Analogs within the imidazo[1,5-alpha]quinoxalin-4-one series had substantially decreased binding affinity as compared to the quinoxaline urea series. These compounds ranged from antagonists to full agonists by in vitro analysis, with several derivatives having roughly 4-fold greater intrinsic activity than diazepam as indicated by Cl- current measurement. Numerous compounds from both series were effective in antagonizing metrazole-induced seizures, consistent with anti-convulsant properties and possible anxiolytic activity. Most of the quinoxaline ureas and quinoxalin-4-ones were active in an acute electroshock physical dependence side effect assay in mice precluding further development.

High-affinity partial agonist imidazo[1,5-a]quinoxaline amides, carbamates, and ureas at the gamma-aminobutyric acid A/benzodiazepine receptor complex.

A series of imidazo[1,5-a]quinoxaline amides, carbamates, and ureas which have high affinity for the gamma-aminobutyric acid A/benzodiazepine receptor complex was developed. Compounds within this class have varying efficacies ranging from antagonists to full agonists. However, most analogs were found to be partial agonists as indicated by [35S]TBPS and Cl- current ratios. Many of these compounds were also effective in antagonizing metrazole-induced seizures in accordance with anticonvulsant and possible anxiolytic activity. Selected quinoxalines displayed limited benzodiazepine-type side effects such as ethanol potentiation and physical dependence in animal models. Dimethylamino urea 41 emerged as the most interesting analog, having a partial agonist profile in vitro while possessing useful activity in animal models of anxiety such as the Vogel and Geller assays. In accordance with its partial agonist profile, 41 was devoid of typical benzodiazepine side effects.

Piperazine imidazo[1,5-a]quinoxaline ureas as high-affinity GABAA ligands of dual functionality.

A series of imidazo[1,5-a]quinoxaline piperazine ureas appended with a tert-butyl ester side chain at the 3-position was developed. Analogues within this series have high affinity for the gamma-aminobutyric acid A (GABAA)/benzodiazepine receptor complex with efficacies ranging from inverse agonists to full agonists. Many analogues were found to be partial agonists as indicated by [35S]TBPS and Cl- current ratios. Uniquely, a number of these analogues were found to have a bell-shaped dose-response profile in the alpha1 beta2 gamma2 subtype as determined by whole cell patch-clamp technique, where in vitro efficacy was found to decrease with increasing drug concentration. Many of the compounds from this series were effective in antagonizing metrazole-induced seizures, consistent with anticonvulsant and possibly anxiolytic activity. Additionally, several analogues were also effective in lowering cGMP levels (to control values) after applied stress, also consistent with anxiolytic-like properties. The most effective compounds in these screens were also active in animal models of anxiety such as the Vogel and Geller assays. The use of the piperazine substituent allowed for excellent drug levels and a long duration of action in the central nervous system for many of the quinoxalines, as determined by ex vivo assay. Pharmacokinetic analysis of several compounds indicated excellent oral bioavailability and a reasonable half-life in rats. From this series emerged two partial agonists (55, 91) which had good activity in anxiolytic models, acceptable pharmacokinetics, and minimal benzodiazepine-type side effects.

Contributions of cysteine 114 of the human D3 dopamine receptor to ligand binding and sensitivity to external oxidizing agents.

1. Cysteine 114 (C114) of the human dopamine D3 receptor is located at the helical face of transmembrane segment III (TMIII) near aspartate 110, a counterion for the amine group of catecholamines. The contributions of C114 to receptor function were investigated here using site-directed mutagenesis of C114 to serine. 2. The C114S mutant, as expressed in Sf-9 cells, bound aminotetralin antagonists (UH-232 and AJ-76) and several agonists ((-)3-PPP, apomorphine, pramipexole and quinpirole) with markedly lower affinities as compared to the wild type D3 receptor, but bound other structurally diverse dopaminergic ligands with only minor changes in affinity. Because an N-propyl substituent is the only common structural feature among most affected ligands, we propose that the mutation alters 'a propyl cleft' on the receptor. The mutation hardly affected quinpirole-dependent [35S]-GTPgammaS binding, suggesting C114 plays a minimal role in receptor-G-protein coupling. 3. N-Ethylmaleimide(NEM), a sulfhydryl modifying agent, blocked ligand binding to the D3 receptor, but not to the C114S mutant. We infer that C114 is the primary residue on the D3 receptor vulnerable to external oxidizing agents. Dopamine D2long and D4(2) receptors contain highly homologous TMIII sequences including an equivalent cysteine residue. However, only the D2long receptor, not the D4(2) receptor, displayed NEM sensitivity similar to that of the D3 receptor. 4. We conclude that C114 is critical for high affinity interactions between the D3 receptor and ligands containing an N-propyl substituent, and unlike its counterpart in the D4(2) receptor, is highly susceptible to external oxidizing agents.

Advantages of heterologous expression of human D2long dopamine receptors in human neuroblastoma SH-SY5Y over human embryonic kidney 293 cells.

The human D2long dopamine receptor when expressed heterologously in a human neuronal cell line, SH-SY5Y, produced more robust functional signals than when expressed in a human embryonic kidney cell line, HEK293. Quinpirole (agonist)-induced GTPgamma(35)S binding and high affinity sites were 3 - 4 fold greater in SH-SY5Y than in HEK293 cells. N-type Ca(2+) channel currents present in SH-SY5Y cells, but not HEK293 cells, were inhibited potently by quinpirole with a half-maximal inhibitory concentration of 0.15+/-0.03 nM. Inhibition of adenylyl cyclases by agonists, on the other hand, was of similar potency and efficacy in the two cell lines. GTPgamma(35)S-Bound Galpha subunits from quinpirole-activated and solubilized membranes were monitored upon immobilization with various Galpha-specific antibodies. Galpha(i) and Galpha(o) subunits were highly labelled with GTPgamma(35)S in SH-SY5Y cells, but only Galpha(i) subunits were labelled in HEK293 cells. The additional G(o) coupling in SH-SY5Y cells could arise, at least in part, from the presence of G(o) coupled-effectors, such as the N-type Ca(2+) channel, and may contribute to robust agonist-induced GTPgamma(35)S binding, which is a reliable means for measuring ligand intrinsic efficacy. It appears that expression of neuronal G protein-coupled receptors in neuronal environments could reveal additional functional characteristics that are absent in non-neuronal cell lines. This appears to be due to, at least in part, to the presence of neuron-specific effectors. These findings underscore the importance of the cellular environment in which drug actions are examined, particularly in the face of intensive efforts to develop drugs for G protein-coupled receptors of various origins.

Comparison of pyrrolidinyl and piperidinyl benzamides for their anticonvulsant activity and inhibitory action on sodium channel.

1. A pair of benzamide analogues containing a pyrrolidinyl or piperidinyl group was examined for their anticonvulsant activity against the electroshock-induced seizures in mice and the ability to block the voltage-gated Na channel in N1E-115 cells, in comparison with the prototype compound, U-54494A, (+/-)-cis-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-ben zam ide , a potent anticonvulsant and a Na channel blocker. 2. The pyrrolidinyl benzamide (U-49524E) was found to be effective against the electroshock-induced seizures (ED50 = 35 mg kg-1, i.p.) whereas the benzamide with a piperidinyl moiety (U-49132E) was inactive (ED50 greater than 100 mg kg-1). 3. Using whole-cell patch clamp techniques we found that U-49132E was several times less potent, with an IC50 of 396 microM as compared to 118 microM for U-49524E at the holding potential of -80 mV, and was much slower in blocking Na channels with a half-time of 10.7 +/- 1.1 min vs. 2.2 +/- 0.4 min for its counterpart. 4. Qualitatively, their general modes of interaction with Na channels were similar to each other and to that of U-54494A in that they interacted with the resting and slowly-inactivated states of the channels and exhibited a use-dependent inhibition because of a slow recovery from the inactivated state in the presence of the drugs. 5. Comparison of their physicochemical properties, shows the less potent and slowly acting U-49132E is more hydrophobic and bulkier than U-49524E, but has the same pKa. This suggests that the drugs approach the Na channel through a narrow and hydrophilic pathway.6. Overall, this study underscores the importance of inhibiting the Na channel to the anticonvulsant activity of the benzamide compounds.

Effects of GABA and various allosteric ligands on TBPS binding to cloned rat GABA(A) receptor subtypes.

1. [35S]t-butylbicyclophosphorothionate (TBPS) is a high affinity ligand for the picrotoxin site of GABA(A) receptors. Here we examined TBPS binding to the cloned receptors made of alpha 1, alpha 3 or alpha 6 in combination with beta 2 or beta 2 and gamma 2 subunits, in the presence of GABA and several allosteric ligands (diazepam, methyl 6,7-dimethoxy-4-methyl-beta-carboline-3-carboxylate (DMCM), 3 alpha,21-dihydroxy-5 alpha-pregnan-20-one (5 alpha-THDOC), pentobarbitone and Zn). The cloned receptors were transiently expressed in SF-9 insect cells by infecting with recombinant baculoviruses. 2. In alpha beta subtypes, GABA at nanomolar concentrations enhanced TBPS binding but inhibited binding at micromolar concentrations. Half maximal GABA concentrations for enhancement or inhibition of TBPS binding were correlated with high and low affinity GABA binding sites, respectively, in individual subtypes. The maximal enhancement of binding also varied according to the alpha isoform (alpha 3 beta 2 >> alpha 1 beta 2). In alpha beta gamma subtypes, TBPS binding was unaffected by GABA at nanomolar concentrations, but was inhibited by GABA at micromolar concentrations. Addition of gamma 2 thus appeared to abolish conformational coupling between high affinity GABA sites and TBPS sites, and also altered low affinity GABA sites; in particular, the half maximal GABA concentration for inhibition of TBPS binding changed from > 100 (alpha 6 beta 2) to 1 microM (alpha 6 beta 2 gamma 2). 3. Allosteric ligands also altered TBPS binding to sensitive GABA(A) receptor subtypes. For instance,diazepam only in the alpha 1 beta2 gamma 2 and alpha 3 beta 2 gamma 2 subtypes, and 5 alpha-THDOC in all the subtypes enhanced TBPS binding in the absence of GABA, and intensified the inhibitory effect of GABA. Pentobarbitone exhibited only the latter effect in all the subtypes we examined.4. DMCM and Zn, inhibitors of GABA-induced Cl currents in alpha beta gamma and alpha beta subtypes, respectively,produced opposite effects to agonists, decreasing TBPS binding in the absence of GABA and attenuating(or eliminating in the case of Zn) the inhibitory effect of GABA on TBPS binding.5. These results show that GABA binding sites and their conformational coupling with TBPS sites are differentially affected by the alpha isoform (particularly alpha 6 as compared to alpha l or alpha 3) and by quaternary interactions involving the gamma 2 subunit. Moreover, changes in TBPS binding by allosteric ligands include not only direct (allosteric) effects on TBPS sites but also indirect effects via GABA sites, and are consistent with their known subtype selectivity and functionality from previous studies.

Alterations of the benzodiazepine site of rat alpha 6 beta 2 gamma 2-GABAA receptor by replacement of several divergent amino-terminal regions with the alpha 1 counterparts.

1. The benzodiazepine site of the alpha 6 beta 2 gamma 2 subtype of gamma-aminobutyric acidA (GABAA) receptors is distinguishable from that of the alpha 1 beta 2 gamma 2 subtype by its inability to interact with classical benzodiazepines (i.e., diazepam) and its agonistic response to Ro 15-1788, which behaves as an antagonist in the alpha 1 beta 2 gamma 2 subtype. 2. The point mutation of Arg 100 of the alpha 6 subunit to histidine (the corresponding residue in alpha 1) has been shown to enable the alpha 6 beta 2 gamma 2 subtype to interact with diazepam but failed in this study to abolish the ability of Ro 15-1788 to enhance GABA-induced Cl- currents. 3. Here we identified the segment of P161 to L187 of alpha 6 to contain the functional region responsible for the agonistic action of Ro 15-1788. Its replacement with the corresponding alpha 1 sequence abolished the ability of Ro 15-1788 to enhance GABA currents without appreciable effects on its binding affinity to the benzodiazepine site or on the functionality of the other benzodiazepine site ligands such as diazepam, U-92330 and 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate. These data support the evidence that the functionality of a given ligand could arise from a single region of the benzodiazepine site, not shared by others. 4. In addition we have learned that several residues in the N-terminal region of alpha 6, such as R100, V142 and N143, have the ability to influence GABA-dependent Cl- current induction probably by allosterically modulating low affinity GABA sites.

Enhancement by GABA of the association rate of picrotoxin and tert-butylbicyclophosphorothionate to the rat cloned alpha 1 beta 2 gamma 2 GABAA receptor subtype.

1. We examined how gamma-aminobutyric acid (GABA) influences interaction of picrotoxin and tert-butylbicyclophosphorothionate (TBPS) with recombinant rat alpha 1 beta 2 gamma 2 GABAA receptors stably expressed in human embryonic kidney cells (HEK293), as monitored with changes in Cl- currents measured by the whole-cell patch clamp technique. 2. During application of GABA (5 microM) for 15 s, picrotoxin and TBPS dose-dependently accelerated the decay of inward GABA-induced currents (a holding potential of -60 mV under a symmetrical Cl- gradient). The drugs, upon preincubation with the receptors, also reduced the initial current amplitude in a preincubation time and concentration-dependent manner. This indicates their interaction with both GABA-bound and resting receptors. 3. The half maximal inhibitory concentration for picrotoxin and TBPS at the beginning of a 15 s GABA (5 microM) pulse was several times greater than that obtained at the end of the pulse. GABA thus appears to enhance picrotoxin and TBPS potency, but only at concentrations leading to occupancy of both high and low affinity GABA sites, i.e., 5 microM. Preincubation of the receptors with the drugs in the presence of GABA at 200 nM, which leads to occupancy of only high affinity GABA sites in the alpha 1 beta 2 gamma 2 subtype, produced no appreciable change in potency of picrotoxin or TBPS. This indicates that they preferentially interact with multiliganded, but not monoliganded receptors, unlike U-93631, a novel ligand to the picrotoxin site, which has higher affinity to both mono- and multiliganded receptors than resting receptors. 4. The time-dependent decay and preincubation time-dependent reduction of initial amplitude of GABA-induced Cl- currents followed monoexponential time courses, and time constants thus obtained displayed a linear relationship with drug concentration. Analysis of the data using a kinetic model with a single drug site showed that GABA (5 microM) enhanced the association rate for picrotoxin and TBPS nearly 100 fold, but their dissociation rate only 10 fold. The dissociation rate obtained from current recovery from picrotoxin or TBPS block yielded nearly identical values to the above analysis.5. We conclude that picrotoxin and TBPS interact with both resting and GABA-bound receptors, but their affinity for the latter is about 10 times greater than that for the former, largely due to a markedly increased association rate to the multiliganded receptors (but not monoliganded ones). This and our earlier study with U-93631 improves our understanding of functional coupling between GABA and picrotoxin sites, which appears to be useful in characterizing the mode of interaction for various picrotoxin site ligands.