Benzodiazepine modulation of the rat GABAA receptor α4ß3γ2L subtype expressed in Xenopus oocytes.

The effects of benzodiazepines on GABA(A) receptors are dependent largely on the particular α subunit isoform that is present in the receptor pentamer. The inclusion of either the α4 or α6 subunit is generally thought to render the receptor insensitive to classical benzodiazepines. We expressed the rat α4ß3γ2L subtype in Xenopus oocytes and observed that both diazepam and flunitrazepam significantly potentiated GABA-gated currents. This potentiation occurred at nanomolar concentrations similar to those seen at the most abundant "diazepam-sensitive" receptor i.e., the α1ß2γ2 subtype. In the α4ß3γ2L receptor, the effects of diazepam and flunitrazepam were inhibited by nanomolar concentrations of the benzodiazepine site antagonists, Ro15-1788 and ZK93426. The presence of the ß3 subunit appears to be important for this modulation since diazepam did not affect GABA responses mediated by recombinant α4ß1γ2L or α4ß2γ2L receptors. Interestingly, when the α4ß3γ2L receptor was expressed in HEK293 cells, diazepam and flunitrazepam displaced the relatively non-selective benzodiazepine site ligand, [(3)H]Ro15-4513, only at high concentrations (>10 µM) demonstrating a lack of high affinity binding for these classical benzodiazepines. Functional studies of the cell-expressed receptors using whole cell recording techniques showed that neither diazepam nor flunitrazepam potentiated GABA-evoked currents although currents were enhanced by nanomolar concentrations of Ro15-4513. These results suggest that the observed benzodiazepine modulation of the α4ß3γ2L subtype depends on the expression system used and may be specific for expression in Xenopus oocytes.

Insights into the structure and pharmacology of GABA(A) receptors.

GABA is the major inhibitory neurotransmitter in the adult mammalian CNS. The ionotropic GABA type A receptors (GABA(A)Rs) belong to the Cys-loop family of receptors. Each member of the family is a large pentameric protein in which each subunit traverses the cell membrane four times. Within this family, the GABA type A receptors are particularly important for their rich pharmacology as they are targets for a range of therapeutically important drugs, including the benzodiazepines, barbiturates, neuroactive steroids and anesthetics. This review discusses new insights into receptor properties that allow us to begin to relate the structure of an individual receptor to its functional and pharmacological properties.

Isomerization of the proline in the M2-M3 linker is not required for activation of the human 5-HT3A receptor.

Each subunit of the cation-selective members of the Cys-loop family of ligand-gated ion channels contains a conserved proline residue in the extracellular loop between the second and third transmembrane domains. In the mouse homomeric 5-hydroxytryptamine type 3A (5-HT(3)A) receptor, the effects of substitution of this proline by unnatural amino acids led to the suggestion that trans-cis isomerization of the protein backbone at this position is integral to agonist-induced channel opening [Nature (2005) vol. 438, pp. 248-252]. We explored the generality of this conclusion using natural amino acid mutagenesis of the homologous human 5-HT(3)A receptor. The conserved proline (P303) was substituted by either a histidine or tryprophan and the mutant receptors were expressed in Xenopus oocytes. These mutations did not significantly affect the magnitude of agonist-mediated currents, compromise channel gating by 5-HT or inhibition of 5-HT-induced currents by either picrotoxin or d-tubocurarine. The mutations did, however, result in altered dependence on extracellular Ca(2+) concentration and a 10-fold increase in the rate of receptor desensitization. These results demonstrate an important role for P303 in 5-HT(3)A receptor function but indicate that trans-cis isomerization at this proline is unlikely to be a general mechanism underlying the gating process.

Bioactive contaminants leach from disposable laboratory plasticware.

Disposable plasticware such as test tubes, pipette tips, and multiwell assay or culture plates are used routinely in most biological research laboratories. Manufacturing of plastics requires the inclusion of numerous chemicals to enhance stability, durability, and performance. Some lubricating (slip) agents, exemplified by oleamide, also occur endogenously in humans and are biologically active, and cationic biocides are included to prevent bacterial colonization of the plastic surface. We demonstrate that these manufacturing agents leach from laboratory plasticware into a standard aqueous buffer, dimethyl sulfoxide, and methanol and can have profound effects on proteins and thus on results from bioassays of protein function. These findings have far-reaching implications for the use of disposable plasticware in biological research.

Epibatidine binds to four sites on the Torpedo nicotinic acetylcholine receptor.

The nicotinic acetylcholine receptor (nAChR) from Torpedo electric organ is a pentamer of homologous subunits. This receptor is generally thought to carry two high affinity sites for agonists under equilibrium conditions. Here we demonstrate directly that each Torpedo nAChR carries at least four binding sites for the potent neuronal nAChR agonist, epibatidine, i.e., twice as many sites as for alpha-bungarotoxin. Using radiolabeled ligand binding techniques, we show that the binding of [(3)H]-(+/-)-epibatidine is heterogeneous and is characterized by two classes of binding sites with equilibrium dissociation constants of about 15nM and 1muM. These classes of sites exist in approximately equal numbers and all [(3)H]-(+/-)-epibatidine binding is competitively displaced by acetylcholine, suberyldicholine and d-tubocurarine. These results provide further evidence for the complexity of agonist binding to the nAChR and underscore the difficulties in determining simple relationships between site occupancy and functional responses.

Atomic force microscopy reveals the stoichiometry and subunit arrangement of the alpha4beta3delta GABA(A) receptor.

The GABA(A) receptor is a chloride-selective ligand-gated ion channel of the Cys-loop superfamily. The receptor consists of five subunits arranged pseudosymmetrically around a central pore. The predominant form of the receptor in the brain contains alpha(1)-, beta(2)-, and gamma(2)-subunits in the arrangement alphabetaalphagammabeta, counter-clockwise around the pore. GABA(A) receptors containing delta-instead of gamma-subunits, although a minor component of the total receptor population, have interesting properties, such as an extrasynaptic location, high sensitivity to GABA, and potential association with conditions such as epilepsy. They are therefore attractive targets for drug development. Here we addressed the subunit arrangement within the alpha(4)beta(3)delta form of the receptor. Different epitope tags were engineered onto the three subunits, and complexes between receptors and anti-epitope antibodies were imaged by atomic force microscopy. Determination of the numbers of receptors doubly decorated by each of the three antibodies revealed a subunit stoichiometry of 2alpha:2beta:1delta. The distributions of angles between pairs of antibodies against the alpha- and beta-subunits both had peaks at around 144 degrees , indicating that these pairs of subunits were nonadjacent. Decoration of the receptor with ligands that bind to the extracellular domain (i.e., the lectin concanavalin A and an antibody that recognizes the beta-subunit N-terminal sequence) showed that the receptor preferentially binds to the mica extracellular face down. Given this orientation, the geometry of complexes of receptors with both an antibody against the delta-subunit and Fab fragments against the alpha-subunits indicates a predominant subunit arrangement of alphabetaalphadeltabeta, counter-clockwise around the pore when viewed from the extracellular space.

Identification of a domain in the delta subunit (S238-V264) of the alpha4beta3delta GABAA receptor that confers high agonist sensitivity.

We have expressed the alpha4beta3delta and alpha4beta3gamma2L subtypes of the rat GABAA receptor in Xenopus oocytes and have investigated their agonist activation properties. GABA was a more potent agonist of the alpha4beta3delta receptor (EC50 approximately 1.4 micromol/L) than of the alpha4beta3gamma2L subtype (EC50 approximately 27.6 micromol/L). Other GABAA receptor agonists (muscimol, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol, imidazole-4-amino acid) displayed similar subtype selectivity. The structural determinants underlying these differences have been investigated by co-expressing chimeric delta/gamma2L subunits with alpha4 and beta3 subunits. A stretch of amino acids in the delta subunit, S238-V264, is shown to play an important role in determining both agonist potency and the efficacies of full or partial agonists. This segment includes transmembrane domain 1 and the short intracellular loop that leads to the second transmembrane domain. The effects of the competitive antagonists, bicuculline and SR95531, and the channel blocker, picrotoxin, were not significantly affected by the incorporation of chimeric subunits. As the delta and gamma2L subunits have not been previously implicated directly in agonist binding, we suggest that the effects are likely to arise from changes in the transduction mechanisms that link agonist binding to channel activation.

Exploring ligand recognition and ion flow in comparative models of the human GABA type A receptor.

We present two comparative models of the GABA(A) receptor. Model 1 is based on the 4-A resolution structure of the nicotinic acetylcholine receptor from Torpedo marmorata and represents the unliganded receptor. Two agonists, GABA and muscimol, two benzodiazepines, flunitrazepam and alprazolam, together with the general anaesthetic halothane, have been docked to this model. The ion flow is also explored in model 1 by evaluating the interaction energy of a chloride ion as it traverses the extracellular, transmembrane and intracellular domains of the protein. Model 2 differs from model 1 only in the extracellular domain and represents the liganded receptor. Comparison between the two models not only allows us to explore commonalities and differences with comparative models of the nicotinic acetylcholine receptor, but also suggests possible protein sub-domain interactions with the GABA(A) receptor not previously addressed.

A single point mutation of the GABA(A) receptor alpha5-subunit confers fluoxetine sensitivity.

Fluoxetine has been reported to be a novel allosteric modulator of GABA(A) receptors with the notable exception of receptors that contain the alpha5-subunit isoform [Robinson, R.T., Drafts, B.C., Fisher, J.L., 2003. Fluoxetine increases GABA(A) receptor activity through a novel modulatory site. J. Pharmacol. Exp. Ther. 304, 978-984]. A mutagenic strategy has been used to investigate the structural basis for the insensitivity of this subunit. An alpha1/alpha5-subunit chimeragenesis approach first demonstrated the importance of the alpha1-subunit N-terminal sequence E165-D183 (corresponding to alpha5 E169-D187) in fluoxetine modulation. Specific amino acid substitutions in this domain subsequently revealed that a single mutation in the alpha5-subunit to the equivalent residue in alpha1 (T179A) was sufficient to confer fluoxetine sensitivity to the alpha5-containing receptor. However, the reciprocal mutation in the alpha1-subunit (A175T) did not result in a loss in sensitivity, suggesting the involvement of additional determinants for fluoxetine modulation. A comparative modeling approach was used to probe amino acids that may lie in close proximity to alpha1A175. This led serendipitously to the identification of a specific residue, alpha1F45, which, when mutated to an alanine, resulted in a significant decrease in potency for activation of the receptor by GABA and also reduced the efficacies of the partial agonists, THIP and P4S.

Chain length dependence of the interactions of bisquaternary ligands with the Torpedo nicotinic acetylcholine receptor.

The interactions of a series of bisholine esters [(CH3)3N+CH(2)CH2OCO-(CH2)n-COOCH2CH2N+(CH3)3] with the Torpedo nicotinic acetylcholine receptor have been investigated. In equilibrium binding studies, [3H]-suberyldicholine (n=6) binds to an equivalent number of sites as [3H]-acetylcholine and with similar affinity (KD approximately 15 nM). In competition studies, all bischoline esters examined displaced both radioligands in an apparently simple competitive manner. Estimated dissociation constants (KI) showed clear chain length dependence. Short chain molecules (n6) had high affinity similar to suberyldicholine. Functional responses were measured by either rapid flux techniques using Torpedo membrane vesicles or voltage-clamp analyses of recombinant receptors expressed in Xenopus oocytes. Both approaches revealed that suberyldicholine (EC50 approximately 3.4 microM) is 14-25-fold more potent than acetylcholine. However, suberyldicholine elicited only about 45% of the maximum response of the natural ligand, i.e., it is a partial agonist. The potency of this bischoline series increased with chain length. Whereas the shorter ligands (nor=4) had similar (or higher) potency to suberyldicholine. Ligand efficacy had an approximately bell-shaped dependence on chain length and compounds where nor=8 were very poor partial agonists. Based on estimates of interonium distances, we suggest that bisquaternary ligands can interact with multiple binding sites on the nAChR and, depending on the conformational state of the receptor, these sites are 15-20A apart.

Tryptophan 86 of the alpha subunit in the Torpedo nicotinic acetylcholine receptor is important for channel activation by the bisquaternary ligand suberyldicholine.

Suberyldicholine, a bisquaternary compound, is a potent nicotinic acetylcholine receptor agonist. Previously, we suggested that at least some of the unusual binding properties of this ligand may be a consequence of its ability to cross-link two binding "subsites" within each of the high-affinity agonist binding domains [Dunn, S. M. J., and Raftery, M. A. (1997) Biochemistry 36, 3846-3853]. Tryptophan 86 of the alpha subunit has previously been implicated in the binding of agonist to this receptor. However, on the basis of the crystal structure of a homologous acetylcholine binding protein, this residue is predicted to lie 15-20 A from the high-affinity site, i.e., a distance that approximates the interonium distance of suberyldicholine. Tryptophan 86 was mutated to either an alanine or a phenylalanine, and the mutated subunit was coexpressed with wild-type beta, gamma, and delta subunits in Xenopus oocytes. Although the alanine mutation resulted in a loss of receptor expression, the alphaW86F mutant receptor was expressed on the oocyte surface, albeit with a much reduced efficiency. Acetylcholine-evoked currents of the alphaW86F receptor were not significantly different from those of the wild type with respect to the concentration dependence of channel activation, receptor desensitization, or d-tubocurarine inhibition. In contrast, the EC(50) for suberyldicholine-mediated activation of the alphaW86F receptor was increased by approximately 500-fold. Furthermore, suberyldicholine-evoked currents in the mutant receptor did not desensitize and were insensitive to block by d-tubocurarine. Thus, tryptophan 86 of the Torpedo receptor alpha subunit may be part of a subsite for recognition of suberyldicholine and other bisquaternary ligands.

Activation of the Torpedo nicotinic acetylcholine receptor. The contribution of residues alphaArg55 and gammaGlu93.

The Torpedo nicotinic acetylcholine receptor is a heteropentamer (alpha2betagammadelta) in which structurally homologous subunits assemble to form a central ion pore. Viewed from the synaptic cleft, the likely arrangement of these subunits is alpha-gamma-alpha-delta-beta lying in an anticlockwise orientation. High affinity binding sites for agonists and competitive antagonists have been localized to the alpha-gamma and alpha-delta subunit interfaces. We investigated the involvement of amino acids lying at an adjacent interface (gamma-alpha) in receptor properties. Recombinant Torpedo receptors, expressed in Xenopus oocytes, were used to investigate the consequences of mutating alphaArg55 and gammaGlu93, residues that are conserved in most species of the peripheral nicotinic receptors. Based on homology modeling, these residues are predicted to lie in close proximity to one another and it has been suggested that they may form a salt bridge in the receptor's three-dimensional structure (Sine et al. 2002 J Biol Chem277, 29 210-29 223). Although substitution of alphaR55 by phenylalanine or tryptophan resulted in approximately a six-fold increase in the EC50 value for acetylcholine activation, the charge reversal mutation (alphaR55E) had no significant effect. In contrast, the replacement of gammaE93 by an arginine conferred an eight-fold increase in the potency for acetylcholine-induced receptor activation. In the receptor carrying the double mutations, alphaR55E-gammaE93R or alphaR55F-gammaE93R, the potency for acetylcholine activation was partially restored to that of the wild-type. The results suggest that, although individually these residues influence receptor activation, direct interactions between them are unlikely to play a major role in the stabilization of different conformational states of the receptor.

Identification of a residue in the gamma-aminobutyric acid type A receptor alpha subunit that differentially affects diazepam-sensitive and -insensitive benzodiazepine site binding.

GABAA receptors that contain either the alpha4- or alpha6-subunit isoform do not recognize classical 1,4-benzodiazepines (BZDs). However, other classes of BZD site ligands, including beta-carbolines, bind to these diazepam-insensitive receptor subtypes. Some beta-carbolines [e.g. ethyl beta-carboline-3-carboxylate (beta-CCE) and methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM)] display a higher affinity for alpha4- compared to alpha6-containing receptors. In order to identify the structural determinants that underlie these affinity differences, we constructed chimeric alpha6/alpha4 subunits and co-expressed these with wild-type rat beta2 and gamma2L subunits in tsA201 cells for radioligand binding analysis. After identification of candidate regions, site-directed mutagenesis was used to narrow the ligand selectivity to a single amino acid residue (alpha6N204/alpha4I203). Substitutions at alpha6N204 did not alter the affinity of the imidazobenzodiazepine Ro15-4513. A homologous mutation in the diazepam-sensitive alpha1 subunit (S205N) resulted in a 7-8-fold reduction in affinity for the beta-carbolines examined. Although the binding of the classical agonist flunitrazepam was relatively unaffected by this mutation in the alpha1 subunit, the affinity for Ro15-1788 and Ro15-4513 was decreased by approximately 19-fold and approximately 38-fold respectively. The importance of this residue, located in the Loop C region of the extracellular N-terminus of the subunit protein, emphasizes the differential interaction of ligands with the alpha subunit in diazepam-sensitive and -insensitive receptors.

Effects of dextroamphetamine, lithium chloride, sodium valproate and carbamazepine on intraplatelet Ca2+ levels.

OBJECTIVE: To explore the possible involvement of second-messenger pathways in the pathophysiology of bipolar disorder and the mechanism of action of mood stabilizers, we investigated the effects of dextroamphetamine (a model for mania) and the most widely used mood stabilizers, lithium chloride, sodium valproate and carbamazepine, on intraplatelet levels of calcium ion ([Ca2+). DESIGN: In the first part of the study, dextroamphetamine was administered in vivo in a double-blind, placebo-controlled, crossover design. In the second part of the study, platelets from untreated subjects were incubated in vitro with dextroamphetamine, lithium chloride, sodium valproate or carbamazepine. PARTICIPANTS: Fifteen healthy men between 18 and 45 years of age. OUTCOME MEASURES: Basal, thrombin-induced and serotonin- (5-HT) induced intraplatelet [Ca2+] determined by means of fura-2 fluorescent intensity. RESULTS: In vivo administration of dextroamphetamine had no effect on basal or agonist-induced intraplatelet [Ca2+]. However, in vitro basal platelet [Ca2+] was significantly higher in samples incubated with dextroamphetamine (86.8 nmol/L [standard error of the mean, SEM, 3.9], p < 0.001), lithium chloride (76.4 nmol/L [SEM 3.1], p < 0.002), sodium valproate (82.7 nmol/L [SEM 3.7], p < 0.001) and carbamazepine (84.8 nmol/L [SEM 3.3], p < 0.001) than in the controls (58.2 nmol/L [SEM 2.3]). Thrombin-induced and 5-HT-induced peak cytosolic [Ca2+] were significantly greater than control levels in samples incubated with carbamazepine (277.1 nmol/L [SEM 19.9] v. 195.8 nmol/L [SEM 12.2], p < 0.002; and 153.0 nmol/L [SEM 8.2] v. 115.4 nmol/L [SEM 5.7], p < 0.003, respectively). CONCLUSIONS: This study does not support the involvement of intraplatelet [Ca2+] in the dextroamphetamine model of mania; however, the modulation of intraplatelet [Ca2+] by the mood stabilizers lithium chloride, sodium valproate and carbamazepine implicates intracellular [Ca2+] in the therapeutic mechanisms of these drugs and the pathophysiological basis of mania.

Comparison of the effects of xenon and halothane on voltage-dependent Ca(2+) fluxes in rabbit T-tubule membranes.

The effects of xenon and halothane on depolarization-induced (45)Ca(2+) fluxes mediated by voltage-dependent Ca(2+) channels were investigated in transverse tubule membrane vesicles from rabbit skeletal muscle. Halothane, in the concentration range of 0.5-2 mM, caused a significant inhibition of (45)Ca(2+) fluxes. Xenon tested in the range of 60%-100% did not affect the (45)Ca(2+) fluxes. Radioligand binding studies indicated that xenon and halothane have different effects on the specific binding of [(3)H]Isradipine to transverse tubule membranes. Halothane caused a significant inhibition on the specific binding of [(3)H]Isradipine. In controls and in presence of 0.5 mM halothane, B(max) values were 26.9 pmole/mg and 15.1 pmole/mg, and K(D) values were 238 pM and 247 pM, respectively. On the other hand, there was no effect of xenon (60%-100%) on the characteristics of [(3)H]Isradipine binding. In conclusion, results indicate that xenon and halothane differ in their effects on the function of voltage-dependent Ca(2+) channels and on the specific binding of [(3)H]Isradipine in skeletal muscle membranes.

Functional consequences of the loss of high affinity agonist binding to gamma-aminobutyric acid type A receptors. Implications for receptor desensitization.

We reported previously that tyrosine 62 of the beta2 subunit of the gamma-aminobutyric acid, type A (GABA(A)) receptor is an important determinant of high affinity agonist binding and that recombinant alpha1beta2gamma2(L) receptors carrying the Y62S mutation lack measurable high affinity sites for [3H]muscimol. We have now examined the effects of disrupting these sites on the macroscopic desensitization properties of receptors expressed in Xenopus oocytes. Desensitization was measured by the ability of low concentrations of bath-perfused agonist to reduce the current responses elicited by subsequent challenges with saturating concentrations of GABA. Wild-type receptors were desensitized by pre-perfused muscimol with an IC50 approximately 0.7 microm, which correlates well with the lower affinity sites for this agonist that are measured in direct binding studies. Receptors carrying the beta2 Y62S and Y62F mutations desensitized at slightly higher (2-7-fold) agonist concentrations. However, at low perfusate concentrations, the Y62S-containing receptor recovered from the desensitized state even in the continued presence of agonist. The characteristics of desensitization in the wild-type and mutant receptors lead us to suggest that the major role of the high affinity agonist-binding site(s) of the GABA(A) receptor is not to induce desensitization but rather to stabilize the desensitized state once it has been formed.

Effects of isoflurane on voltage-dependent calcium fluxes in rabbit T-tubule membranes: comparison with alcohols.

The effects of racemic (+/-) and (+)- and (-)-stereoisomers of isoflurane on depolarization-induced (45)Ca(2+) fluxes mediated by voltage-dependent Ca(2+) channels were investigated in transverse tubule membrane vesicles from rabbit skeletal muscle. In the concentration range 0.5 to 2 mM, (+/-)-isoflurane inhibited (45)Ca(2+) fluxes and functionally modulated the effects of the Ca(2+) channel antagonist nifedipine (1-10 microM). Isoflurane-induced inhibition of (45)Ca(2+) fluxes was not significantly affected by pretreatment with either pertussis toxin (5 microg/ml) or phorbol 12-myristate 13-acetate (50 nM). Further experiments indicated that there were no significant differences between (+)- and (-)-stereoisomers of isoflurane with respect to the extent of inhibition of (45)Ca(2+) fluxes. Radioligand binding studies indicated that racemic and (+)- and (-)-isoflurane were equally effective in displacing the specific binding of [(3)H]PN 200-110 to transverse tubule membranes. There were no apparent differences between the effects of (+)- and (-)-isoflurane on the characteristics of [(3)H]PN 200-110 binding. Although the concentrations of isoflurane for the inhibitions of (45)Ca(2+) fluxes and radioligand bindings were similar, the concentrations of n-alcohols required for the inhibition of (45)Ca(2+) fluxes were lower than those for the displacement of radioligand. Comparison of the data for the displacement of [(3)H]PN 200-110 binding and the inhibition of (45)Ca(2+) fluxes by isoflurane and by n-alcohols suggested that both isoflurane and n-alcohols may have more than a single binding site. In conclusion, results indicate that isoflurane, independent of intracellular Ca(2+) levels, nonstereospecifically inhibits the function of voltage-dependent Ca(2+) channels and this effect is mediated through multiple binding sites.