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PubMed2003

Homology modeling of the transmembrane domain of the human calcium sensing receptor and localization of an allosteric binding site.

A homology model for the human calcium sensing receptor (hCaR) transmembrane domain utilizing bovine rhodopsin (bRho) structural information was derived and tested by docking the allosteric antagonist, NPS 2143, followed by mutagenesis of predicted contact sites. Mutation of residues Phe-668 (helix II), Arg-680, or Phe-684 (helix III) to Ala (or Val or Leu) and Glu-837 (helix VII) to Ile (or Gln) reduced the inhibitory effects of NPS 2143 on [Ca2+]i responses. The calcimimetic NPS R-568 increases the potency of Ca2+ in functional assays of CaR. Mutations at Phe-668, Phe-684, or Glu-837 attenuated the effects of this compound, but mutations at Arg-680 had no effect. In all cases, mutant CaRs responded normally to Ca2+ or phenylalanine, which act at distinct site(s). Discrimination by the Arg-680 mutant is consistent with the structural differences between NPS 2143, which contains an alkyl bridge hydroxyl group, and NPS R-568, which does not. The homology model of the CaR transmembrane domain robustly accounts for binding of both an allosteric antagonist and agonist, which share a common site, and provides a basis for the development of more specific and/or potent allosteric modulators of CaR. These studies suggest that the bRho backbone can be used as a starting point for homology modeling of even distantly related G protein-coupled receptors and provide a rational framework for investigation of the contributions of the transmembrane domain to CaR function.

Allosteric Site↗
PubMed2006

Differential coupling of alpha7 and non-alpha7 nicotinic acetylcholine receptors to calcium-induced calcium release and voltage-operated calcium channels in PC12 cells.

Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-gated cation channels that can modulate various neuronal processes by altering intracellular Ca(2+) levels. Following nAChR stimulation Ca(2+) can enter cells either directly, through the intrinsic ion channel, or indirectly following voltage-operated Ca(2+) channel (VOCC) activation; Ca(2+) levels can subsequently be amplified via Ca(2+)-induced Ca(2+) release from intracellular stores. We have used subtype-selective nAChR agonists to investigate the Ca(2+) sources contributing to alpha7 and non-alpha7 nAChR-mediated increases in intracellular Ca(2+) in PC12 cells. Application of the alpha7 nAChR positive allosteric modulator PNU 120596 (10 mum), in conjunction with the alpha7 nAChR agonist, compound A [(R)-N-(1-azabicyclo[2.2.2]oct-3-yl)(5-(2-pyridyl)thiophene-2-carboxamide), 10 nm], produces a rapid increase in fluo-3 fluorescence that is prevented by the selective alpha7 nAChR antagonist alpha-bungarotoxin. The non-alpha7 nAChR agonist 5-Iodo-A-85380 produces alpha-bungarotoxin-insensitive increases in intracellular Ca(2+) (EC(50) = 11.2 mum). Using these selective agonists or KCl in conjunction with general and selective VOCC inhibitors, we demonstrate that the primary route of Ca(2+) entry following either non-alpha7 nAChR activation or KCl stimulation is via L-type VOCCs. In contrast, the alpha7 nAChR-mediated response is unaffected by VOCC blockers but is inhibited by modulators of intracellular Ca(2+) stores. These results indicate that alpha7 and non-alpha7 nAChRs are differentially coupled to Ca(2+)-induced Ca(2+) release and VOCCs, respectively.

Animals↗
PubMed1997

Modulation of nicotinic ACh-, GABAA- and 5-HT3-receptor functions by external H-7, a protein kinase inhibitor, in rat sensory neurones.

1. The effects of external H-7, a potent protein kinase inhibitor, on the responses mediated by gamma-aminobutyric acid A type (GABAA)-, nicotinic acetylcholine (nicotinic ACh)-, ionotropic 5-hydroxytryptamine (5-HT3)-, adenosine 5'-triphosphate (ATP)-, N-methyl-D-aspartate (NMDA)- and kainate (KA)-receptors were studied in freshly dissociated rat dorsal root ganglion neurone by use of whole cell patch-clamp technique. 2. External H-7 (1-1000 microM) produced a reversible, dose-dependent inhibition of whole cell currents activated by GABA, ACh and 5-HT. 3. Whole-cell currents evoked by ATP, 2-methylthio-ATP, NMDA and KA were insensitive to external H-7. 4. External H-7 shifted the dose-response curve of GABA-activated currents downward without changing the EC50 significantly (from 15.0 +/- 4.0 microM to 18.0 +/- 5.0 microM). The maximum response to GABA was depressed by 34.0 +/- 5.3%. This inhibitory action of H-7 was voltage-independent. 5. Intracellular application of H-7 (20 microM), cyclic AMP (1 mM) and BAPTA (10 mM) could not reverse the H-7 inhibition of GABA-activated currents. 6. The results suggest that external H-7 selectively and allosterically modulates the functions of GABAA-, nicotine ACh- and 5-HT3 receptors via a common conserved site in the external domain of these receptors.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed2006

DANGER, a novel regulatory protein of inositol 1,4,5-trisphosphate-receptor activity.

We report the cloning and characterization of DANGER, a novel protein which physiologically binds to inositol 1,4,5-trisphosphate receptors (IP(3)R). DANGER is a membrane-associated protein predicted to contain a partial MAB-21 domain. It is expressed in a wide variety of neuronal cell lineages where it localizes to membranes in the cell periphery together with IP(3)R. DANGER interacts with IP(3)R in vitro and co-immunoprecipitates with IP(3)R from cellular preparations. DANGER robustly enhances Ca(2+)-mediated inhibition of IP(3) RCa(2+) release without affecting IP(3) binding in microsomal assays and inhibits gating in single-channel recordings of IP(3)R. DANGER appears to allosterically modulate the sensitivity of IP(3) RtoCa(2+) inhibition, which likely alters IP(3)R-mediated Ca(2+) dynamics in cells where DANGER and IP(3)R are co-expressed.

Allosteric Site↗
PubMed2000

Effects of treatment with GABA(A) receptor subunit antisense oligodeoxynucleotides on GABA-stimulated 36Cl- influx in the rat cerebral cortex.

GABA(A) receptor function was studied in cerebral cortical vesicles prepared from rats after intracerebroventricular microinjections of antisense oligodeoxynucleotides (aODNs) for alpha1, gamma2, beta1, beta2 subunits. GABA(A) receptor alpha1 subunit aODNs decreased alpha1 subunit mRNA by 59+/-10%. Specific [3H]GABA binding was decreased by alpha1 or beta2 subunit aODNs (to 63+/-3% and 64+/-9%, respectively) but not changed by gamma2 subunit aODNs (94+/-5%). Specific [3H]flunitrazepam binding was increased by alpha1 or beta2 subunit aODNs (122+/-8% and 126+/-11%, respectively) and decreased by gamma2 subunit aODNs (50+/-13%). The "knockdown" of specific subunits of the GABA(A )receptor significantly influenced GABA-stimulated 36Cl- influx. Injection of alpha1 subunit aODNs decreased basal 36Cl- influx and the GABA Emax; enhanced GABA modulation by diazepam; and decreased antagonism of GABA activity by bicuculline. Injection of gamma2 subunit aODNs increased the GABA Emax; reversed the modulatory efficacy of diazepam from enhancement to inhibition of GABA-stimulation; and reduced the antagonist effect of bicuculline. Injection of beta2 subunit aODNs reduced the effect of diazepam whereas treatment with beta1 subunit aODNs had no effect on the drugs studied. Conclusions from our studies are: (1) alpha1 subunits promote, beta2 subunits maintain, and gamma2 subunits suppress GABA stimulation of 36Cl- influx; (2) alpha1 subunits suppress, whereas beta2, and gamma2 subunits promote allosteric modulation by benzodiazepines; (3) diazepam can act as an agonist or inverse agonist depending on the relative composition of the receptor subunits: and (4) the mixed competitive/non-competitive effects of bicuculline result from activity at alpha1 and gamma2 subunits and the lack of activity at beta1 and beta2 subunits.

Animals↗
PubMed1990

Effect of brief treatment at alkaline pH on the properties of UDP-glucuronosyltransferase.

The kinetic properties of UDP-glucuronosyltransferase were measured after brief treatment of liver microsomes at alkaline pH, followed by assay with p-nitro-phenol as aglycone, at pH 7.5. Enzyme activity increased in a graded fashion as the pH of pretreatment was increased above 8.0, with apparent maximal activation of eight-fold for a pretreatment pH of 11.1. The pH for half maximal activation was 10.6. Brief treatment at alkaline pH prior to assay at pH 7.5 was associated too with a graded conversion of the kinetics of the enzyme from non-Michaelis-Menten to Michaelis-Menten at pH 11.7. Sensitivity to the allosteric modulator, UDP-N-acetylglucosamine decreased as the pH increased. A fifty percent loss of sensitivity to UDP-N-acetylglucosamine-induced activation occurred at pH 10.6. Thus, pretreatment at alkaline pH had irreversible effects on the properties of UDP-glucuronosyltransferase in microsomes. In order to establish the cause for the irreversibility of the changes induced by alkaline pH, microsomes were treated at pH 11.6 prior to purifying UDP-glucuronosyltransferase. Enzyme purified from alkali-treated and untreated microsomes had approximately the same specific activity. More importantly, responses to activation by lipids, and regeneration of allosteric properties were the same for both purified enzymes (from alkali-treated and control microsomes). Pure enzyme was not activated by pretreatment at alkaline pH. We interpret these data to mean that the irreversible effects of alkaline pH on the properties of UDP-glucuronosyltransferase in microsomes were not due to direct effects on the enzyme, but to how the enzyme interacted normally with molecules within the plane of the membrane.

Animals↗
PubMed2005

The therapeutic potential of drugs that target cannabinoid receptors or modulate the tissue levels or actions of endocannabinoids.

There are at least 2 types of cannabinoid receptor, CB(1) and CB(2), both G protein coupled. CB(1) receptors are expressed predominantly at nerve terminals and mediate inhibition of transmitter release, whereas CB(2) receptors are found mainly on immune cells, their roles including the modulation of cytokine release and of immune cell migration. Endogenous agonists for cannabinoid receptors also exist. These "endocannabinoids" are synthesized on demand and removed from their sites of action by cellular uptake and intracellular enzymic hydrolysis. Endocannabinoids and their receptors together constitute the endocannabinoid system. This review summarizes evidence that there are certain central and peripheral disorders in which increases take place in the release of endocannabinoids onto their receptors and/or in the density or coupling efficiency of these receptors and that this upregulation is protective in some disorders but can have undesirable consequences in others. It also considers therapeutic strategies by which this upregulation might be modulated to clinical advantage. These strategies include the administration of (1) a CB(1) and/or CB(2) receptor agonist or antagonist that does or does not readily cross the blood brain barrier; (2) a CB(1) and/or CB(2) receptor agonist intrathecally or directly to some other site outside the brain; (3) a partial CB(1) and/or CB(2) receptor agonist rather than a full agonist; (4) a CB(1) and/or CB(2) receptor agonist together with a noncannabinoid, for example, morphine or codeine; (5) an inhibitor or activator of endocannabinoid biosynthesis, cellular uptake, or metabolism; (6) an allosteric modulator of the CB(1) receptor; and (7) a CB(2) receptor inverse agonist.

Animals↗
PubMed1992

Density of 1,4-dihydropyridine receptors decreases in the hearts of aging hamsters.

To determine whether density and properties of myocardial voltage-sensitive calcium channels may change in the hearts of aged animals, we have compared specific binding of [3H]-nitrendipine ([3H]-NTP) to 1,4-dihydropyridine (DHP) receptors in ventricular homogenate and membrane fractions prepared from the hearts of adult hamsters of different ages. In crude homogenate preparations from young adult animals (70 days) the density (Bmax) of [3H]-NTP binding was 82 +/- 6 fmol/mg protein (n = 5 hearts). By contrast, Bmax values for 400-, 500- and 600-day-old animals were 54 +/- 5, 59 +/- 11 and 50 +/- 2 fmol/mg protein, respectively (significantly lower than control, P = 0.004, n = 4-5 hearts/group). The affinity (Kd) of [3H]-NTP binding was not affected by increasing age. Similar results were obtained in a partially purified membrane fraction. Allosteric modulation of [3H]-NTP binding by the phenylalkylamine, verapamil and the benzothiazepine, diltiazem were preserved in aging heart. These studies demonstrate that the density of myocardial DHP receptors declines in the aged hamster, although the properties of these receptors are unchanged. This suggests that a decrease in voltage-sensitive calcium channels may be associated with normal myocardial aging.

Aging↗
PubMed1995

Coordination of Ca2+ by the pore region glutamates is essential for high-affinity dihydropyridine binding to the cardiac Ca2+ channel alpha 1 subunit.

The molecular determinants for Ca2+ modulation of dihydropyridine (DHP) binding to cardiac Ca2+ channels were identified by mutational neutralization of the glutamate residues that comprise the Ca2+ channel selectivity filter. The binding activity of the DHP (+)-[3H]isradipine, monitored after expression of wild-type and mutant alpha 1 subunits in COS-7 cells, was markedly reduced in four single mutants and a double mutant. Evidence for decreased Ca2+ affinity was obtained for two single mutants in kinetic and equilibrium binding studies. Mutational destabilization of Ca2+ binding resulted in a concomitant decrease of (+)-[3H]isradipine binding affinity. Recovery of (+)-[3H]isradipine binding activity by the allosteric modulator (+)-tetrandrine in two single mutants was associated with a recovery of Ca2+ and DHP binding kinetics to wild-type values. Our findings demonstrate that high-affinity DHP binding is dependent on Ca2+ coordination by glutamate residues which form the selectivity filter of the channel pore.

Alkaloids↗
PubMed2002

Effects of S-nitrosation and cross-linking of hemoglobin on hypoxic pulmonary vasoconstriction in isolated rat lungs.

Free hemoglobin (Hb) and red blood cells augment hypoxic pulmonary vasoconstriction (HPV) by scavenging nitric oxide (NO). S-nitrosation of Hb (SNO-Hb) may confer vasodilatory properties by allowing release of NO during deoxygenation and/or by interaction with small-molecular weight thiols. Likewise, cross-linking of free Hb may limit its vasoconstrictive effect by preventing abluminal movement of the molecule. We compared the effects of free SNO-Hb and Hb intramolecularly cross-linked at the beta-cysteine 93 residue [Bis(maleidophenyl)-polyethylene glycol2000HbA (Bis-Mal-PEGHb)] to those of free oxyHb on pulmonary artery pressure (PAP), HPV, and exhaled NO (eNO) in isolated, perfused rat lungs. Ventilation of lungs with anoxic gas for 5 minutes reduced perfusate PO2 to 11+/-1.0 Torr. Addition of SNO-Hb or Bis-Mal-PEGHb (100 micromol/L) to buffer perfusate increased normoxic PAP and augmented HPV in similar magnitude as free oxyHb, but had no effect on eNO. Addition of the allosteric modulator inositol hexaphosphate to increase Hb P50 and the thiol glutathione (GSH) to allow removal of NO from Hb via transnitrosation to the perfusate did not reduce augmentation of HPV by SNO-Hb or increase eNO. GSH resulted in an approximately 50% reduction in perfusate [S-nitrosothiol], in association with an increase in perfusate [metHb]. Free SNO-Hb is a net NO scavenger and pulmonary vasoconstrictor in this model, although thiol-mediated release of NO from SNO-Hb does occur. However, release of NO from SNO-Hb was not influenced by deoxygenation-mediated allosteric changes in Hb across a broad range of oxyHb saturation. Cross-linking of Hb does not limit its pulmonary vasoconstrictor effects.

Animals↗
PubMed2005

Selective anxiolysis produced by ocinaplon, a GABA(A) receptor modulator.

Benzodiazepines remain widely used for the treatment of anxiety disorders despite prominent, often limiting side effects including sedation, muscle relaxation, and ataxia. A compound producing a robust anxiolytic action comparable to benzodiazepines, but lacking these limiting side effects at therapeutic doses (an anxioselective agent), would represent an important advance in the treatment of generalized anxiety disorder, and perhaps other anxiety disorders. Here we report that the pyrazolo[1,5-a]-pyrimidine, ocinaplon, exhibits an anxioselective profile in both preclinical procedures and in patients with generalized anxiety disorder, the most common of the anxiety disorders. In rats, ocinaplon produces significant muscle relaxation, ataxia, and sedation only at doses >25-fold higher than the minimum effective dose (3.1 mg/kg) in the Vogel "conflict" test. This anticonflict effect is blocked by flumazenil (Ro 15-1788), indicating that like benzodiazepines, ocinaplon produces an anxiolytic action through allosteric modulation of GABA(A) receptors. Nonetheless, in eight recombinant GABA(A) receptor isoforms expressed in Xenopus oocytes, the potency and efficacy of ocinaplon to potentiate GABA responses varied with subunit composition not only in an absolute sense, but also relative to the prototypical benzodiazepine, diazepam. In a double blind, placebo controlled clinical trial, a 2-week regimen of ocinaplon (total daily dose of 180-240 mg) produced statistically significant reductions in the Hamilton rating scale for anxiety scores. In this study, the incidence of benzodiazepine-like side effects (e.g., sedation, dizziness) in ocinaplon-treated patients did not differ from placebo. These findings indicate that ocinaplon represents a unique approach both for the treatment and understanding of anxiety disorders.

Adult↗
PubMed2002

A model of the human M2 muscarinic acetylcholine receptor.

The M2 muscarinic acetylcholine receptor belongs to the family of rhodopsin like G-Protein Coupled Receptors. This subtype of muscarinic receptors is of special interest because it bears, aside from an orthosteric binding site, also an allosteric binding site. Based on the X-ray structure of bovine rhodopsin a complete homology model of the human M2 receptor was developed. For the orthosteric binding site point mutations and binding studies with different agonists and antagonists are available. This knowledge was utilized for an initial verification of the M2 model. Allosteric modulation of activity is mediated by structurally different ligands such as gallamine, caracurine V salts or W84 (a hexamethonium-derivative). Caracurine V derivatives with different affinities to M2 were docked using GRID-fields. Subsequent molecular dynamics simulations yielded different binding energies based on diverse electrostatic and lipophilic interactions. The calculated affinities are in good agreement to experimentally determined affinities.

Allosteric Site↗
PubMed1999

Glutathione and signal transduction in the mammalian CNS.

The tripeptide glutathione (GSH) has been thoroughly investigated in relation to its role as antioxidant and free radical scavenger. In recent years, novel actions of GSH in the nervous system have also been described, suggesting that GSH may serve additionally both as a neuromodulator and as a neurotransmitter. In the present article, we describe our studies to explore further a potential role of GSH as neuromodulator/neurotransmitter. These studies have used a combination of methods, including radioligand binding, synaptic release and uptake assays, and electrophysiological recording. We report here the characteristics of GSH binding sites, the interrelationship of GSH with the NMDA receptor, and the effects of GSH on neural activity. Our results demonstrate that GSH binds via its gamma-glutamyl moiety to ionotropic glutamate receptors. At micromolar concentrations GSH displaces excitatory agonists, acting to halt their physiological actions on target neurons. At millimolar concentrations, GSH, acting through its free cysteinyl thiol group, modulates the redox site of NMDA receptors. As such modulation has been shown to increase NMDA receptor channel currents, this action may play a significant role in normal and abnormal synaptic activity. In addition, GSH in the nanomolar to micromolar range binds to at least two populations of binding sites that appear to be distinct from all known excitatory amino acid receptor subtypes. GSH bound to these sites is not displaceable by glutamatergic agonists or antagonists. These binding sites, which we believe to be distinct receptor populations, appear to recognize the cysteinyl moiety of the GSH molecule. Like NMDA receptors, the GSH binding sites possess a coagonist site(s) for allosteric modulation. Furthermore, they appear to be linked to sodium ionophores, an interpretation supported by field potential recordings in rat cerebral cortex that reveal a dose-dependent depolarization to applied GSH that is blocked by the absence of sodium but not by lowering calcium or by NMDA or (S)-2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate antagonists. The present data support a reevaluation of the role of GSH in the nervous system in which GSH may be involved both directly and indirectly in synaptic transmission. A full accounting of the actions of GSH may lead to more comprehensive understanding of synaptic function in normal and disease states.

Animals↗
PubMed2005

New therapeutic frontiers for metabotropic glutamate receptors.

Our understanding of glutamatergic transmission in the central nervous system has been greatly expanded with the discovery and investigation of the metabotropic glutamate receptor family. Complementing the ionotropic glutamate-gated ion channels, these G-protein coupled receptors play critical roles in neuronal and glial functions such as the modulation of neuronal excitability, synaptic transmission, and various metabolic functions. Because of the ubiquitous distribution of glutamatergic synapses, it has been deemed likely that mGlu receptors participate in most, if not all, major functions of the CNS. It is predicted that the wide diversity and heterogeneous distribution of mGlu receptor subtypes will provide avenues to develop clinically relevant pharmacological agents that target specific CNS systems. mGlu receptors are regulated by differences in expression, alternative splicing patterns, and interactions with other proteins in the cell and it is anticipated that an understanding of these modifiers of mGlu receptor function will open new opportunities for pharmacological tool development and new therapeutic strategies. Over the past decade, an increasing number of selective agonists, antagonists, and allosteric modulators have been developed which target distinct mGlu receptor subtypes; many of these agents have now been further validated in numerous electrophysiological and behavioral models. The combination of these pharmacological tools, in conjunction with genetic approaches, has led to major advances in our understanding of the roles of mGlu receptors in the regulation of CNS function and animal behavior. These studies suggest the exciting possibility that drugs active at mGlu receptors will be useful in treatment of a wide variety of neurological and psychiatric disorders such as Parkinson's disease, anxiety disorders, and schizophrenia.

Allosteric Regulation↗
PubMed2000

Subunit composition and pharmacological characterization of gamma-aminobutyric acid type A receptors in frog pituitary melanotrophs.

The frog pars intermedia is composed of a single population of endocrine cells directly innervated by gamma-aminobutyric acid (GABA)ergic nerve terminals. We have previously shown that GABA, acting through GABA(A) receptors, modulates both the electrical and secretory activities of frog pituitary melanotrophs. The aim of the present study was to take advantage of the frog melanotroph model to determine the relationship between the subunit composition and the pharmacological properties of native GABA(A) receptors. Immunohistochemical labeling revealed that in situ and in cell culture, frog melanotrophs were intensely stained with alpha2-, alpha3-, gamma2-, and gamma3-subunit antisera and weakly stained with a gamma1-subunit antiserum. Melanotrophs were also immunolabeled with a monoclonal antibody to the beta2/beta3-subunit. In contrast, frog melanotrophs were not immunoreactive for the alpha1-, alpha5-, and alpha6-isoforms. The effects of allosteric modulators of the GABA(A) receptor on GABA-activated chloride current were tested using the patch-clamp technique. Among the ligands acting at the benzodiazepine-binding site, clonazepam (EC50, 5 x 10(-9) M), diazepam (EC50, 10(-8) M), zolpidem (EC50, 3 x 10(-8) M), and beta-carboline-3-carboxylic acid methyl ester (EC50, 10(-6) M) were found to potentiate the whole cell GABA-evoked current in a dose-dependent manner. Methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (IC50, 3 x 10(-5) M) inhibited the current, whereas Ro15-4513 had no effect. Among the ligands acting at other modulatory sites, etomidate (EC50, 2 x 10(-6) M) enhanced the GABA-evoked current, whereas 4'-chlorodiazepam (IC50, 4 x 10(-7) M), ZnCl2 (IC50, >5 x 10(-5) M), and furosemide (IC50, >3 x 10(-4) M) depressed the response to GABA. PK 11195 did not affect the GABA-evoked current or its inhibition by 4'-chlorodiazepam. The results indicate that the native GABA(A) receptors in frog melanotrophs are formed by combinations of alpha2-, alpha3-, beta2/3-, gamma1-, gamma2-, and gamma3-subunits. The data also demonstrate that clonazepam is the most potent, and zolpidem is the most efficient positive modulator of the native receptors. Among the inhibitors, 4'-chlorodiazepam is the most potent, whereas ZnCl2 is the most efficient negative modulator of the GABA(A) receptors. The present study provides the first correlation between subunit composition and the functional properties of native GABA(A) receptors in nontumoral endocrine cells.

Animals↗
PubMed2005

Effects on heart rate of an anti-M2 acetylcholine receptor immune response in mice.

Autoantibodies in vitro modulating the M2 acetylcholine receptor (M2ACh-R) were observed in patients with idiopathic dilated cardiomyopathy (IDC) or Chagas' cardiomyopathy (ChC). We investigated the in vivo consequences on heart rate of such antibodies in mice immunized with a peptide derived from the second extracellular loop of the M2ACh-R compared with mice immunized with an irrelevant peptide. Sera of mice immunized with the M2ACh-R-derived peptide recognized the M2ACh-R on immunoblots and enhanced agonist activity of carbachol toward the M2AChR transfected in CHO cells. In vivo, no difference could be shown in heart rate or heart rate variability between the two groups of mice. The decrease in heart rate induced by carbachol was more pronounced, however, in the M2ACh-R immunized mice. The increase in heart rate induced by atropine, gallamine, and isoproterenol was significantly attenuated in the M2ACh-R immunized mice. Analysis of heart rate variability further argued for an increased parasympathetic response to different drugs in the M2ACh-R immunized mice. Antibodies raised against the M2AChR can behave as positive M2AChR allosteric modulators in vivo. They might be protective in boosting the activity of the parasympathetic drive to the heart, especially in patients with a high sympathetic tone.

Allosteric Regulation↗
PubMed1992

Effect of a benzodiazepine (chlordiazepoxide) on a GABAA receptor from rat brain. Requirement of only one bound GABA molecule for channel opening.

Chlordiazepoxide (CDPX) enhanced the rate of chloride exchange mediated by the major GABAA receptor found on sealed native membrane vesicles from rat cerebral cortex. The initial rate constant for chloride exchange for this receptor, (JA), a measure of open channel, was determined from the progress of GABA-mediated influx of 36Cl-. The dependence of JA on GABA concentration was hyperbolic in the presence of CDPX (150 microM, sufficient to give maximum enhancement of chloride exchange rate) but sigmoid in its absence. Enhancement of channel opening (10-fold at 0.3 microM GABA) decreased with increasing GABA concentration. The maximal response, above 1,000 microM GABA, was unaltered. The half-response concentration was reduced from 80 microM to 50 microM. CDPX alone caused no measurable 36Cl- exchange. In the presence of CDPX, channel opening occurred with only one bound GABA molecule, whereas in its absence, channel opening with two bound GABA molecules was much more favorable. This could not be direct allosteric modulation of the channel opening conformational change by binding of CDPX at effector sites, but could be explained by an additional change of the receptor on binding CDPX to give a closed state which gave channel opening mediated by a single GABA binding site. Another possibility is that CDPX could act at one of the channel opening binding sites without a postulated, second closed conformational state.

Allosteric Regulation↗
PubMed2004

Inverse agonism and neutral antagonism at cannabinoid CB1 receptors.

There are at least two types of cannabinoid receptor, CB1 and CB2, both G protein coupled. CB1 receptors are expressed predominantly at nerve terminals and mediate inhibition of transmitter release whereas CB2 receptors are found mainly on immune cells, one of their roles being to modulate cytokine release. Endogenous cannabinoid receptor agonists also exist and these "endocannabinoids" together with their receptors constitute the "endocannabinoid system". These discoveries were followed by the development of a number of CB1- and CB2-selective antagonists that in some CB1 or CB2 receptor-containing systems also produce "inverse cannabimimetic effects", effects opposite in direction from those produced by cannabinoid receptor agonists. This review focuses on the CB1-selective antagonists, SR141716A, AM251, AM281 and LY320135, and discusses possible mechanisms by which these ligands produce their inverse effects: (1) competitive surmountable antagonism at CB1 receptors of endogenously released endocannabinoids, (2) inverse agonism resulting from negative, possibly allosteric, modulation of the constitutive activity of CB1 receptors in which CB1 receptors are shifted from a constitutively active "on" state to one or more constitutively inactive "off" states and (3) CB1 receptor-independent mechanisms, for example antagonism of endogenously released adenosine at A1 receptors. Recently developed neutral competitive CB1 receptor antagonists, which are expected to produce inverse effects through antagonism of endogenously released endocannabinoids but not by modulating CB1 receptor constitutive activity, are also discussed. So too are possible clinical consequences of the production of inverse cannabimimetic effects, there being convincing evidence that released endocannabinoids can have "autoprotective" roles.

Animals↗