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PubMed2002

Wellcome Prize Lecture. Cell surface, ion-sensing receptors.

Changes in extracellular calcium (Ca(2+)o) concentration ([Ca2+]o) affect kidney function both under basal and hormone-stimulated conditions. The molecular identification of an extracellular Ca(2+)-sensing receptor (CaR) has confirmed a direct role of Ca(2+)o on parathyroid and kidney function (i.e. independent of calciotropic hormones) as a modulator of Ca2+ homeostasis. In addition, evidence accumulated over the last 10 years has shown that CaR is also expressed in regions outside the calcium homeostatic system where its role is largely undefined but seems to be linked to regulation of local ionic homeostasis. The parathyroid and kidney CaRs are 1081 and 1079 amino acids long, respectively, and belong to the type III family of G protein-coupled receptors (GPCRs), which includes other CaRs, metabotropic glutamate receptors and putative vomeronasal organ receptors. For the CaR, its low (millimolar) affinity for Ca2+, its positive cooperativity and its large ion-sensing extracellular domain, indicate that the receptor is more sensitive to changes in net cationic charge rather than to a specific ligand. Mg2+, trivalent cations of the lanthanide series and polyvalent cations such as spermine and aminoglycoside antibiotics can all activate the receptor in vitro with EC50 values in the micromolar range for trivalent and polyvalent cations or in the millimolar range for Ca2+ and Mg2+. In addition to true CaR agonists, CaR sensitivity to Ca(2+)o is also susceptible to allosteric modulation by ionic strength, L-amino acids and by pharmacological agents. This review will address endogenous and exogenous CaR agonists, the role of the receptor in the calcium homeostatic system and some speculation on possible role(s) of the CaR in regions not involved in mineral ion homeostasis.

Awards and Prizes↗
PubMed1994

Stanozolol and danazol, unlike natural androgens, interact with the low affinity glucocorticoid-binding sites from male rat liver microsomes.

Some 17 alpha-alkylated androgens used as anabolic agents, such as stanozolol (ST) and danazol (DA), have specific effects on the liver that are not exerted by testosterone. This gives rise to the possibility that a steroid-binding protein, other than the androgen receptor, could modulate the intracellular actions of these agents. Male rat liver microsomes contain a homogeneous population of [3H]dexamethasone ([3H]DEX)-binding sites which we have denominated low affinity glucocorticoid-binding sites (LAGS). Because glucocorticoids, progestagens, and the synthetic estrogen ethynyl estradiol compete with [3H]DEX for binding to the LAGS, we aimed to study the possible interactions between androgens and the LAGS. To investigate whether several androgens had the capability of interacting with the LAGS, we performed competition experiments. The LAGS had no affinity for testosterone or methyltrienolone (R1881). However, some 17 alpha-alkylated androgens (DA (IC50, 116 nM) > ST >> fluoxymesterone > mestaline > methandriol >> methandrostenolone > methyltestosterone) were able to compete with [3H]DEX binding to liver microsomes. ST and DA were potent inhibitors of [3H]DEX binding to liver microsomes. They decreased both the affinity and the number of [3H]DEX-binding sites, increased the dissociation rate of [3H]DEX from the LAGS, and provoked a time- and dose-dependent inactivation of the [3H]DEX-binding site. These results strongly suggest that ST and DA exert a negative allosteric modulation on [3H]DEX binding to the LAGS. The in vivo administration of ST (but not other androgens) to male rats provoked a time- and dose-dependent decrease in the LAGS level. Full recovery of the LAGS concentration required at least 8 h and was blocked by protein synthesis inhibitors. Such results suggest that ST irreversibly inactivates the [3H]DEX-binding site in vivo as it does in vitro. Taken together, these observations are indicative of an irreversible interaction between some 17 alpha-alkylated androgens and the LAGS both in vitro and in vivo and suggest that ST may be an important pharmacological tool that can be used in the elucidation of the molecular structure of the LAGS. These results also mean that the LAGS are a steroid-binding entity able to distinguish between natural androgens and 17 alpha-alkylated testosterone derivatives used as anabolic agents.

Animals↗
PubMed1997

The effects of inhibitors of GABAergic transmission and stress on brain and plasma allopregnanolone concentrations.

1. This study was undertaken to investigate the relationship between a reduction in brain GABAA receptor function and the cerebro-cortical content of 3 alpha-hydroxy-5 alpha-pregnan-20 one (allopregnanolone, AP), a potent endogenous positive modulator of 7-aminobutyric acid (GABA) action at GABAA receptors, with anticonflict and anticonvulsant effects in rodents. 2. An acute depletion of the cerebral content of GABA or an attenuation of GABAA receptor-mediated transmission by systemic injections of isoniazid (375 mg kg-1, s.c.) or FG 7142 (15 mg kg-1, i.p.) induced a transient increase in the cerebro-cortical and plasma concentrations of AP in handling-habituated (not stressed) rats. 3. Two stress paradigms, handling in naive rats and mild foot shock in handling-habituated rats, that reduce central GABAergic tone mimicked the effects of isoniazid and FG 7142 on cortical AP content; foot shock in handling-habituated rats, but not handling in naive animals, also increased plasma AP. Isoniazid, FG 7142, and foot shock also each increased the concentrations of the AP precursors, pregnenolone and progesterone, in both brain and plasma of handling-habituated rats, whereas handling in naive rats increased the concentrations of these steroids only in brain. 4. Pretreatment of handling-habituated rats with the anxiolytic beta-carboline derivative abecarnil, a positive allosteric modulator of GABAA receptors, which per se failed to affect the AP concentration in brain or plasma, prevented the increase in brain and plasma AP induced by foot shock or isoniazid. 5. In adrenalectomized and castrated rats foot shock or isoniazid failed to increase AP both in brain cortex and plasma. 6. These observations indicate that inhibition of GABAergic transmission, induced by foot shock or pharmacological manipulations, results in an increase in the concentrations of AP in brain and plasma, possibly via a modulation of hypothalamic-pituitary-adrenal (HPA) axis. 7. Given that AP enhances GABAA receptor function with high efficacy and potency, an increase in brain AP concentration may be important in the fine tuning of the GABA-mediated inhibitory transmission in the central nervous system.

Adrenalectomy↗
PubMed2000

Binding of Zn2+ to a Ca2+ loop allosterically attenuates the activity of factor VIIa and reduces its affinity for tissue factor.

The protease domain of coagulation factor VIIa (FVIIa) is homologous to trypsin with a similar active site architecture. The catalytic function of FVIIa is regulated by allosteric modulations induced by binding of divalent metal ions and the cofactor tissue factor (TF). To further elucidate the mechanisms behind these transformations, the effects of Zn2+ binding to FVIIa in the free form and in complex with TF were investigated. Equilibrium dialysis suggested that two Zn2+ bind with high affinity to FVIIa outside the N-terminal gamma-carboxyglutamic acid (Gla) domain. Binding of Zn2+ to FVIIa, which was influenced by the presence of Ca2+, resulted in decreased amidolytic activity and slightly reduced affinity for TF. After binding to TF, FVIIa was less susceptible to zinc inhibition. Alanine substitutions for either of two histidine residues unique for FVIIa, His216, and His257, produced FVIIa variants with decreased sensitivity to Zn2+ inhibition. A search for putative Zn2+ binding sites in the crystal structure of the FVIIa protease domain was performed by Grid calculations. We identified a pair of Zn2+ binding sites in the Glu210-Glu220 Ca2+ binding loop adjacent to the so-called activation domain canonical to serine proteases. Based on our results, we propose a model that describes the conformational changes underlying the Zn2+-mediated allosteric down-regulation of FVIIa's activity.

Alanine↗
PubMed1992

Ontogeny of glycine-enhanced [3H]MK-801 binding to N-methyl-D-aspartate receptor-coupled ion channels.

The N-methyl-D-aspartate (NMDA) subtype of glutamate receptor is thought to play a critical role in neuronal development, differentiation and plasticity. A number of studies have shown an enhanced sensitivity to NMDA receptor ligands in neonatal animals. This study examined the ontogenetic changes in the glycinergic modulation of NMDA-coupled cation channels in the developing central nervous system of rat pups. The nonequilibrium binding of the specific channel ligand [3H]MK-801 was used as a measure of NMDA channel access. Glycine (10(-5) M) enhancement of [3H]MK-801 binding at 2 h in forebrain membranes from adult rats was significantly greater than that observed in tissues from 8- to 28-day-old rat pups. This difference was due to changes in the efficacy, but not potency of glycine. The observed ontogenetic changes in the efficacy of glycine-enhanced [3H]MK-801 binding were attributable to developmental changes in receptor site density, as determined by equilibrium [3H]MK-801 saturation isotherms. Kinetic studies revealed that glycine increased the association rate constants of [3H]MK-801 in 8-day and adult membranes by a similar magnitude (0.111 +/- 0.021 vs 0.094 +/- 0.009 nM-1 h-1, respectively). Similarly, the fractional amount of [3H]MK-801 bound (i.e., amount bound at time t normalized to amount bound at equilibrium) in the presence of glycine was relatively constant throughout neonatal development. These findings suggest that the allosteric modulation of the NMDA ionophore by glycine is similar in postnatal and adult rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

[Concurrence of multiple and integrated mechanisms in the modulation of enzyme activities: significance for the regulation of metabolic fluxes].

The activity of some enzymes in a given metabolic pathway is modulated through multiple mechanisms, which operate in a simultaneous and coherent way to produce either stimulation or inhibition. The operation of these mechanisms is illustrated with several enzymes involved in glucose metabolism, by choosing examples from the presentations at the Symposium. Thus the reciprocal interactions of the regulatory mechanisms acting upon hexokinase D ('glucokinase'), phosphofructokinase, fructose 1,6-bisphosphatase and pyruvate kinase were discussed, as well as their relationships with the induction of enzyme conformational changes. In addition, the effects of covalent interconversions on glutamine synthetase activity were briefly analyzed. An outstanding feature exhibited by all these enzymes is the display of a great number of elasticity coefficients, which are differential quotients measuring the dependence of enzymatic activity on each variable that modulates it. A general assumption is that these enzymes make an important contribution to the control of the metabolic flux in which they participate. The flux control, however, appears to be shared in different degrees by all the components of the system, and may be quantified through the differential quotient denominated control coefficient. Some of the problems that emerge in any attempt to estimate these coefficients in the living cells are discussed. The problems derive partly from the complex subcellular structure, the formation of functional compartments resulting from reversible association of the enzymes, one to another and to different cellular components, and the actual state of cell water. These problems make that the results obtained with purified and highly diluted enzymes in most enzymological studies should not be extrapolated directly to what happens in vivo, without a careful evaluation of each particular case. The regulatory role of enzyme activity of fructose 2,6-bisphosphate and its eventual participation as an intermediary in the hormonal control of glycolytic and gluconeogenic fluxes are emphasized. The regulation of yeast fructose 1,6-bisphosphate activity is discussed in relation to the eventual role of allosteric modulators and covalents interconversions as signals for the initiation of intracellular degradation of the enzyme during catabolic inactivation.

Chromatography, DEAE-Cellulose↗
PubMed1992

Modulation of (+)-[3H]pentazocine binding to guinea pig cerebellum by divalent cations.

The ability of cations to modulate the binding of the sigma 1 receptor-selective ligand (+)-[3H]pentazocine to guinea pig cerebellum was investigated. Di- and trivalent cations biphasically inhibited (+)-[3H]pentazocine binding, revealing multiple affinity states. The rank order of potency of these cations (based on the high affinity component of inhibition) was Zn2+ > Co2+ >> La3+ = Ni2+ = Cd2+ = Mn2+ = Gd2+ > Ba2+ = Sr2+ >> Mg2+ > Ca2+. The inhibition of 1,3-[3H]di(2-tolyl)guanidine binding to the sigma 2 receptor by these cations differed qualitatively and quantitatively from their effects on (+)-[3H]pentazocine binding. Although monovalent cations decreased the Kd for (+)-[3H]pentazocine binding, divalent cations split (+)-[3H]pentazocine binding into low and high affinity components. The Bmax of the high affinity component decreased with increasing divalent cation concentrations. Both mono- and divalent cations significantly reduced the rate of association of (+)-[3H]pentazocine with the sigma 1 receptor without altering the dissociation rate. (+)-[3H]Pentazocine binding was not altered by guanine nucleotides or by treatment with cholera or pertussis toxins. However, nonselective cation channel blockers (cinnarizine, hydroxyzine, prenylamine, amiodarone, and proadifen) potently inhibited (+)-[3H]pentazocine binding. These results indicate that physiologically relevant concentrations of divalent cations allosterically modulate (+)-[3H]pentazocine binding to the sigma 1 receptor, to reveal multiple affinity states. These sites do not represent sigma 1 to sigma 2 subtype interconversion or ternary complex formation with guanine nucleotide-binding proteins. However, the rank order of cation potency and the inhibition of binding by cation channel blockers is consistent with a potential role for sigma receptors as constituents of cation channels.

Animals↗
PubMed1996

Neurosteroid modulation of the benzodiazepine receptor in developing chick optic lobe.

The modulation of [3H]flunitrazepam binding by 3 alpha-hydroxy-5 alpha-dihydroprogesterone (3 alpha-OH-DHP) and 3 alpha, 5 alpha-tetrahydrodeoxicorticosterone (3 alpha-THDOC) was investigated in synaptic membranes isolated from chick optic lobe at three developmental stages, in order to evaluate the role of neurosteroids in central nervous system functional maturation. It was demonstrated that both steroids modulate [3H]flunitrazepam binding in a concentration-dependent manner at embryonic day 14, hatching and adult stage, producing maximal [3H]flunitrazepam binding enhancement at early stages of development and declining thereafter. EC50 values for 3 alpha-OH-DHP were lower than for 3 alpha-THDOC at all stages examined. On the other hand, Emax values were higher with 3 alpha-OH-DHP than with 3 alpha-THDOC. Scatchard analysis performed at embryonic day 14, hatching and adult stage showed that dissociation constants (Kd) were of 22.23 +/- 0.15, 18.38 +/- 1.15 and 19.86 +/- 0.62 nM, and maximal number of binding sites (Bmax) were 1.95 +/- 0.15, 3.13 +/- 0.21 and 2.25 +/- 0.13 pmol/mg protein, respectively. By adding 4 microM of either 3 alpha-OH-DHP or 3 alpha-THDOC, Kd values decreased significantly to 10.65 +/- 0.62, 9.71 +/- 0.85 and 13.25 +/- 0.74 nM or 9.54 +/- 0.65, 11.20 +/- 1.27 and 12.96 +/- 1.38 nM, at the above mentioned stages, respectively. Thus, either drug at the given concentration increased the affinity of benzodiazepine receptor sites for [3H]flunitrazepam, while the density of receptor sites remained unchanged. Our results suggest that these steroids display a positive allosteric modulation of benzodiazepine receptor sites which is inversely related to age.

Animals↗
PubMed2000

Interactions of alcuronium, TMB-8, and other allosteric ligands with muscarinic acetylcholine receptors: studies with chimeric receptors.

A series of ligands that allosterically modulate the binding of classical ligands to muscarinic receptors was evaluated at wild-type and chimeric receptors. All of the ligands studied had highest affinity toward the M(2) subtype and lowest affinity toward the M(5) subtype. The chimeric receptors were mostly M(5) sequence; the amount of M(2) sequence ranged from about 6 to just under 30%. Alcuronium and TMB-8 had much higher affinity for the chimeric receptor that included the M(2) second outer loop of the receptor plus flanking regions of TM4 and TM5 than for any of the other chimeric receptors (the affinities of which remained similar to that of the M(5) subtype). However, this chimera retained the negative cooperativity between alcuronium and the classical antagonist N-methylscopolamine that is characteristic of M(5) (these ligands are positively cooperative at M(2)). Verapamil, tetrahydroaminoacridine, and d-tubocurarine were also sensitive to that chimeric substitution, although verapamil and tetrahydroaminoacridine had even higher affinity for a chimera with M(2) sequence in TM7. None of these ligands shared gallamine's sensitivity to a region of the third outer loop, but studies in which obidoxime reversed the allosteric effects of gallamine and other ligands suggested that they nevertheless compete for a common site. In summary, although the present data are consistent with previous studies that have suggested that allosteric ligands bind to the outermost regions of muscarinic receptors, it appears that different allosteric ligands may derive subtype selectivity from different regions of the receptor.

Acetylcholine↗
PubMed2006

Histamine augments beta2-adrenoceptor-induced cyclic AMP accumulation in human prostate cancer cells DU-145 independently of known histamine receptors.

Androgen-independent prostate cancer cells DU-145 express a number of G protein-coupled receptors, including histamine H1 receptors. There is evidence for the presence of beta-adrenoceptors in the human prostate, and in this work we set out to characterise the expression of beta-adrenoceptors by DU-145 cells, their linking to cyclic AMP (cAMP) formation and the possible modulation by histamine H1 receptors of beta-adrenoceptor function. Saturation [3H]-dihydroalprenolol binding indicated that DU-145 cells express moderate levels of beta-adrenoceptors (22.7+/-2.5 fmol/mg protein), which belong to the beta2-subtype as assessed by inhibition by the antagonists ICI-118,551 and CGP-20712A. Inhibition of [3H]-dihydroalprenolol binding by agonists (noradrenaline, adrenaline and isoproterenol) showed the presence of both high-(53-59%) and low-affinity binding sites. beta-Adrenoceptor stimulation with isoproterenol resulted in robust [3H]-cAMP accumulation (10-30-fold of basal, EC50 142 nM; pEC50 6.85+/-0.05). While not having effect of its own on basal [3H]-cAMP accumulation, histamine significantly augmented the beta2-adrenoceptor-induced response (overall effect 152+/-6% of isoproterenol alone) with EC50 1.35 microM (pEC50 5.87+/-0.06). This effect was independent of extracellular Ca2+, insensitive to antagonists/agonists at H1, H2 or H3/H4 receptors and mimicked by drugs containing an imidazole ring in their chemical structure and by imidazole itself. Taken together, our results show that in DU-145 cells histamine augments beta2-adrenoceptor-induced cAMP independently of the activation of known histamine receptors. The effect may involve other mechanisms such as allosteric modulation of beta2-adrenoceptors by the imidazole moiety of histamine.

Cell Line, Tumor↗
PubMed2005

A novel calcium-sensing receptor antagonist transiently stimulates parathyroid hormone secretion in vivo.

Circulating calcium (Ca(2+)) is a primary regulator of bone homeostasis through its action on PTH secretion. Extracellular Ca(2+) modulates PTH secretion through a cell surface G protein-coupled receptor, the calcium-sensing receptor (CaR). The expression of the CaR suggests a critical role in cellular regulation by calcium in various organs, including parathyroid gland, bone, and kidney. Despite an obvious pharmacological utility for CaR antagonists in the treatment of disease, only a limited number of such classes of compounds exist. We have identified a novel class of small molecules with specific activity at the CaR. This class of compounds is represented by compound 1. It possesses potent antagonist activity at the human CaR with IC(50) values of 64 nm and 230 nm in inhibiting intracellular Ca(2+) flux and inositol phosphate generation in vitro, respectively. When administered to male rats in vivo, compound 1 robustly increased serum PTH levels. The stimulation of PTH secretion was rapid and transient when administered either iv or orally. The pharmacokinetic profile of compound 1 after oral administration revealed that maximal plasma levels of compound were reached within 1 h and the half-life of the compound to be approximately 2 h in rats. These data describe a representative compound of a novel chemical class than previously described allosteric modulators that offer a new avenue for the development of improved treatments of osteoporosis.

Animals↗
PubMed2000

Distress vocalizations in maternally separated mouse pups: modulation via 5-HT(1A), 5-HT(1B) and GABA(A) receptors.

RATIONALE: Young rodents emit ultrasonic vocalizations (USVs) when separated from their dams and littermates. Pharmacological agents that act on GABA(A) and/or 5-HT receptors and that alleviate anxiety in humans reduce the emission of these calls. OBJECTIVES: 1) to investigate specific 5-HT1 receptor subtypes that modulate maternal separation-induced USVs in mice; 2) to assess the behavioral specificity of these effects; and 3) to compare 5-HT1 agonists with a positive neurosteroid modulator of the GABA(A) receptor complex. METHODS: Seven-day old CFW mouse pups were isolated from their littermates and placed onto a 20 degrees C surface for 4 min. USVs between 30 and 80 kHz, grid crossing, and rectal temperature were measured in separate groups of mouse pups following subcutaneous administration of 5-HT1A and 5-HT1B receptor agonists and antagonists, the neurosteroid allopregnanolone, or the benzodiazepine midazolam. RESULTS: The 5-HT1A agonists (+)8-OH-DPAT (0.01-0.1 mg/kg) and flesinoxan (0.3-1.0 mg/kg), the selective 5-HT1B agonist CP-94,253 (0.03-30.0 mg/kg), and the mixed 5-HT1B/2C receptor agonist TFMPP (0.1-10.0 mg/kg) dose-dependently reduced USVs. These effects were reversed by the 5-HT1A receptor antagonist WAY 100,635 (0.1 mg/kg) or the 5-HT1B/D receptor antagonist GR 127935 (0.1 mg/kg). The effects of TFMPP were biphasic; low doses (i.e. 0.01 and 0.03 mg/kg) increased the rate of vocalization. Midazolam and allopregnanolone also reduced USVs. The highest doses of flesinoxan, (+)8-OH-DPAT, and allopregnanolone suppressed locomotion, whereas CP-94,253, TFMPP, and midazolam stimulated motor activity. CONCLUSIONS: These experiments confirm that agonists at the 5-HT1 receptors and a positive allosteric modulator of the GABA(A) receptor complex decrease maternal separation-induced USVs in mice, with 5-HT1B manipulations dissociating the effects on vocalizations from sedative effects.

Animals↗
PubMed2006

A model for agonism and antagonism in an ancient and ubiquitous cAMP-binding domain.

The cAMP-binding domain (CBD) is an ancient and conserved regulatory motif that allosterically modulates the function of a group of diverse proteins, thereby translating the cAMP signal into a controlled biological response. The main receptor for cAMP in mammals is the ubiquitous regulatory (R) subunit of protein kinase A. Despite the recognized significant potential for pharmacological applications of CBDs, currently only one group of competitive inhibitor antagonists is known: the (R(p))-cAMPS family of phosphorothioate cAMP analogs, in which the equatorial exocyclic oxygen of cAMP is replaced by sulfur. It is also known that the diastereoisomer (S(p))-cAMPS with opposite phosphorous chirality is a cAMP agonist, but the molecular mechanism of action of these analogs is currently not fully understood. Previous crystallographic and unfolding investigations point to the enhanced CBD dynamics as a key determinant of antagonism. Here, we investigate the (R(p))- and (S(p))-cAMPS-bound states of R(CBD-A) using a comparative NMR approach that reveals a clear chemical shift and dynamic NMR signature, differentiating the (S(p))-cAMPS agonist from the (R(p))-cAMPS antagonist. Based on these data, we have proposed a model for the (R(p)/S(p))-cAMPS antagonism and agonism in terms of steric and electronic effects on two main allosteric relay sites, Ile(163) and Asp(170), respectively, affecting the stability of a ternary inhibitory complex formed by the effector ligand, the regulatory and the catalytic subunits of protein kinase A. The proposed model not only rationalizes the existing data on the phosphorothioate analogs, but it will also facilitate the design of novel cAMP antagonists and agonists.

Allosteric Site↗
PubMed1996

Protracted treatment with diazepam reduces benzodiazepine1 receptor-mediated potentiation of gamma-aminobutyric acid-induced currents in dissociated rat hippocampal neurons.

Tolerance to benzodiazepines (BZs) is thought to involve alterations of the gamma-aminobutyric acid (GABA)A receptor as a result of the prolonged occupancy of its modulatory BZ recognition site. We used the whole-cell patch-clamp technique to compare the functional and pharmacological properties of GABAA receptors in acutely dissociated hippocampal neurons from the control or diazepam-tolerant rats. Administration of diazepam (15 mg/kg p.o.) twice a day for 10 days induced tolerance as demonstrated by the decreased potency of acute diazepam i.p. injections to protect against pentylenetetrazole-induced clonictonic convulsions (10.5% of tolerant rats protected by 0.1 mg/kg of diazepam against 55% of nontreated rats, 48 hr after the last dose of the chronic treatment). The specific current induced by 1 microM GABA in acutely dissociated hippocampal neurons 48 hr after withdrawal (10.5 +/- 1.3 microA/cm2) was similar to that observed in the control rats (8.7 +/- 0.8 microA/cm2). The EC50 value for GABA was unchanged by the chronic treatment [6.3 (5.4-7.1) and 7.5 (6.2-8.7) microM in neurons from the control and treated rats, respectively]. The potency of the nonselective allosteric modulator diazepam to stimulate Cl- currents was identical in cells from treated rats [EC50 values of 25 (20-30) and 34 (26-41) nM in the control and treated rats, respectively; P < .05], but the potency of the selective BZ1-site ligand zolpidem was decreased [EC50 values of 99 (88-111) and 267 (221-313) nM in the control and treated rats, respectively; P < .05]. The maximal potentiation of the GABA-induced current was significantly decreased with diazepam (maximal potentiation: 168.0 +/- 16.2 and 124.0 +/- 8.9% in the control and treated rats, respectively). These results suggest that tolerance to diazepam is accompanied in hippocampal neurons by a decrease in BZ1 binding sites and in the functional coupling of BZ/GABA recognition sites.

Allosteric Regulation↗
PubMed1997

A point mutation in the gamma2 subunit of gamma-aminobutyric acid type A receptors results in altered benzodiazepine binding site specificity.

Benzodiazepines allosterically modulate gamma-aminobutyric acid (GABA) evoked chloride currents of gamma-aminobutyric acid type A (GABAA) receptors. Coexpression of either rat gamma2 or gamma3, in combination with alpha1 and beta2 subunits, results both in receptors displaying high [3H]Ro 15-1788 affinity. However, receptors containing a gamma3 subunit display a 178-fold reduced affinity to zolpidem as compared with gamma2-containing receptors. Eight chimeras between gamma2 and gamma3 were constructed followed by nine different point mutations in gamma2, each to the homologous amino acid residue found in gamma3. Chimeric or mutant gamma subunits were coexpressed with alpha1 and beta2 in human embryonic kidney 293 cells to localize amino acid residues responsible for the reduced zolpidem affinity. Substitution of a methionine-to-leucine at position 130 of gamma2 (gamma2M130L) resulted in a 51-fold reduction in zolpidem affinity whereas the affinity to [3H]Ro 15-1788 remained unchanged. The affinity for diazepam was only decreased by about 2-fold. The same mutation resulted in a 9-fold increase in Cl 218872 affinity. A second mutation (gamma2M57I) was found to reduce zolpidem affinity by about 4-fold. Wild-type and gamma2M130L-containing receptors were functionally expressed in Xenopus oocytes. Upon mutation allosteric coupling between agonist and modulatory sites is preserved. Dose-response curves for zolpidem and for diazepam showed that the zolpidem but not the diazepam apparent affinity is drastically reduced. The apparent GABA affinity is not significantly affected by the gamma2M130L mutation. The identified amino acid residues may define part of the benzodiazepine binding pocket of GABAA receptors. As the modulatory site in the GABAA receptor is homologous to the GABA site, and to all agonist sites of related receptors, gamma2M130 may either point to a homologous region important for agonist binding in all receptors or define a new region not underlying this principle.

Animals↗
PubMed1993

Establishment of a cellular assay system for G protein-linked receptors: coupling of human NK2 and 5-HT2 receptors to phospholipase C activates a luciferase reporter gene.

A functional cellular assay system was developed for the detection of substances modulating the activity of G protein-coupled receptors, linked to the phospholipase C second messenger system. The human adenocarcinoma cell line A549 was transformed with the Photinus pyralis luciferase gene under the control of the ICAM-1 gene 5'regulatory region and, subsequently, stably transfected with the human neurokinin 2 (NK2) receptor gene. The ICAM-1 promoter is known to be inducible via the phospholipase C signal transduction pathway. In this NK2 receptor test cell line, expression of luciferase was inducible by neurokinin A and other NK2-specific agonists. The order of potency of the three neurokinins substance P, neurokinin A and neuromedin K was consistent with published data and results from ligand binding studies performed with the same NK2 test cell line. The agonistic effect of neurokinin A could be inhibited in a dose-dependent manner by simultaneous addition of NK2-specific antagonists or protein kinase C-inhibitors. Similarly, a stable test cell line expressing the human serotonin 2 receptor was established. Agonist-induced luciferase expression in this cell line was abolished in the presence of 5-HT2-specific antagonists. These cellular assay systems can be employed for the identification of competitive, non-competitive and allosteric modulators of the NK2 and the 5-HT2 receptor, and they represent prototypes for analogous test cell lines for other phospholipase C-coupled receptors.

Biological Assay↗
PubMed2000

Site-directed mutagenesis of the regulatory domain of Escherichia coli carbamoyl phosphate synthetase identifies crucial residues for allosteric regulation and for transduction of the regulatory signals.

Carbamoyl phosphate (CP), the essential precursor of pyrimidines and arginine, is made in Escherichia coli by a single carbamoyl phosphate synthetase (CPS) consisting of 41.4 and 117.7 kDa subunits, which is feed-back inhibited by UMP and activated by IMP and ornithine. The large subunit catalyzes CP synthesis from ammonia in three steps, and binds the effectors in its 15 kDa C-terminal domain. Fifteen site-directed mutations were introduced in 13 residues of this domain to investigate the mechanism of allosteric modulation by UMP and IMP. Two mutations, K993A and V994A, decreased significantly or abolished enzyme activity, apparently by interfering with the step of carbamate synthesis, and one mutation, T974A, negatively affected ornithine activation. S948A, K954A, T974A, K993A and K993W/H995A abolished or greatly hampered IMP activation and UMP inhibition as well as the binding of both effectors, monitored using photoaffinity labeling and ultracentrifugation binding assays. V994A also decreased significantly IMP and UMP binding. L990A, V991A, H995A, G997A and G1008A had more modest effects or affected more the modulation by and the binding of one than of the other nucleotide. K993W, R1020A, R1021A and K1061A were without substantial effects. The results confirm the independence of the regulatory and catalytic centers, and also confirm functional predictions based on the X-ray structure of an IMP-CPS complex. They prove that the inhibitor UMP and the activator IMP bind in the same site, and exclude that the previously observed binding of ornithine and glutamine in this site were relevant for enzyme activation. K993 and V994 appear to be involved in the transmission of the regulatory signals triggered by UMP and IMP binding. These effectors possibly change the position of K993 and V994, and alter the intermolecular contacts mediated by the regulatory domain.

Allosteric Regulation↗
PubMed2001

Evidence for distinct conformations of the two alpha 1 subunits in diazepam-bound GABA(A) receptors.

Benzodiazepines allosterically modulate GABA(A) receptors to increase currents induced by submaximal GABA concentrations. Benzodiazepine-induced conformational changes in the transmembrane domain increase the reactivity of cysteines substituted for a subset of residues in the alpha(1) subunit M3 membrane-spanning segment. With the cysteine-substitution mutant alpha(1)F296Cbeta(1)gamma(2) we previously noted that p-chloromercuribenzenesulfonate (pCMBS(-)) modification in the presence of diazepam potentiated subsequent GABA-induced currents. In contrast, pCMBS(-) modification in the presence of GABA caused inhibition of subsequent responses. We now show that in the presence of diazepam, pCMBS(-) only reacts with the engineered cysteine in one of the two alpha subunits; whereas, in the presence of GABA, pCMBS(-) reacts with the cysteine in the other alpha subunit, or with both cysteines. This implies that the two alpha subunits have distinct conformations in the diazepam-bound state. Based on analysis of single channel kinetic data, others have hypothesized that diazepam only alters the GABA affinity of one of the two GABA binding sites. The results presented here provide structural evidence to support the hypothesis that diazepam binding only alters the conformation of one of the two alpha subunits in a GABA(A) receptor and provides new insights into the mechanism of allosteric potentiation by benzodiazepines.

Allosteric Regulation↗