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PubMed2006

Point mutations in the transmembrane region of GABAB2 facilitate activation by the positive modulator N,N'-dicyclopentyl-2-methylsulfanyl-5-nitro-pyrimidine-4,6-diamine (GS39783) in the absence of the GABAB1 subunit.

GABA(B) receptors are heterodimers of two subunits, GABA(B1) (GB1) and GABA(B2) (GB2). Agonists such as GABA and baclofen bind to the GB1 subunit only, whereas GB2 is essential for G protein activation. Positive allosteric modulators enhance the potency and efficacy of agonists at GABA(B) receptors and are of particular interest because they lack the sedative and muscle relaxant properties of agonists. In this study, we aimed to characterize the interaction of the positive modulator N,N'-dicyclopentyl-2-methylsulfanyl-5-nitro-pyrimidine-4,6-diamine (GS39783) with the GABA(B) receptor heterodimer. Using functional guanosine 5'-O-(3-[(35)S]thio)triphosphate binding assays, we observed positive modulation by GS39783 in different vertebrate species but not in Drosophila melanogaster. However, coexpression of D. melanogaster GB1 with rat GB2 yielded functional receptors positively modulated by GS39783. Together with data from rat/D. melanogaster GB2 subunit chimeras, this pointed to a critical role of the GB2 transmembrane region for positive modulation. We further characterized GS39783 function using point mutations. GS39783 positively modulated GABA responses but also showed considerable agonistic activity at heterodimers containing a mutant rat GB2 subunit with three amino acid substitutions in transmembrane domain VI. It was surprising that in contrast to wild-type rat GB2, this mutant subunit was also activated by GS39783 when expressed without GB1. The mutations of both G706T and A708P are necessary and sufficient for activation and identify a key region for the effect of GS39783 in the GB2 transmembrane region. Our data show that mutations of specific amino acids in GB2 can induce agonism in addition to positive modulation and facilitate GB2 activation in the absence of GB1.

Amino Acid Sequence↗
PubMed1988

Allosteric equilibria in the binding of fibrinogen to platelets.

The binding of fibrinogen to platelets occurs according to the law of mass action. The platelet receptor binds reversibly a single fibrinogen molecule and undergoes a conformational transition between two allosteric states, T and R, that differ in their affinity for fibrinogen. The equilibrium between the two forms is shifted by ADP toward the R (high-affinity) state, thus promoting the aggregation process. This model opens the way to consideration of allosteric modulation of the binding of fibrinogen to its platelet receptor.

Adenosine Diphosphate↗
PubMed2001

Modulation of GABA(A) receptors by benzodiazepines and barbiturates is autonomous of PKC activation.

Previous studies have suggested that activation of calcium-phospholipid-dependent protein kinase (PKC) enhances benzodiazepine (BZD)- and pentobarbital (PB)- mediated potentiation of alpha(1)beta(1)gamma(2) GABA(A) receptors (GABA(A)-Rs). To delineate the underlying mechanism(s), voltage-clamp recordings were performed on recombinant alpha(1)beta(1)gamma(2) GABA(A) receptors functionally expressed in Xenopus laevis oocytes. GABA(A)-Rs were tested for their sensitivity to diazepam and PB before and after incubation in phorbol 12-myristate 13-acetate (PMA). PMA (25 nM) significantly attenuated the GABA(A) current (p<0.05, n=12-19) up to 90%. PMA treatment, however, did not alter the sensitivity to diazepam or pentobarbital. Similar results were obtained with recombinant alpha(1)beta(2)gamma(2) GABA receptors. These data suggest that PKC activation does not alter the allosteric modulation of GABA(A)-Rs by benzodiazepines and barbiturates and is consistent with the observation from other studies in oocytes that PMA decreases the amplitude of the GABA-activated currents via receptor internalization rather than modification of receptor kinetics.

Allosteric Regulation↗
PubMed1996

Astroglia-released factor with negative allosteric modulatory properties at the GABA A receptor.

We have previously shown, using whole-cell patch-clamp techniques, that astrocytes release a negative allosteric modulator of the gamma-aminobutyric acid type A receptor (GABAA receptor) with beta-carboline-like properties, thus, likely to act at the benzodiazepine site. Here, using patch-clamp and binding techniques, we confirm that the low-molecular-weight fraction of astroglia-conditioned medium (ACM lmf) contains a factor(s) that negatively modulates GABAA-receptor function. This factor, like beta-carbolines, enhances the specific binding of [35S]t-butyl bicyclophosphorothionate (TBPS) to adult rat cortical membranes in the presence of GABA. However, it fails to interact with various ligands of the benzodiazepine (BZD) site of the GABAA receptor ([3H]flunitrazepam, [3H]Ro 15-1788 and [3H]Ro 15-4513). The question of the actual binding site of the astroglia-derived factor on the GABAA receptor, thus, remains open and can be addressed only after the purification of the active molecule(s) of ACM Imf has been completed, and a labeled form of the endogenous ligand becomes available. Taken together, however, the data suggest that type 1 astrocytes are able to modulate the effects of the main inhibitory neurotransmission in the central nervous system.

Allosteric Regulation↗
PubMed1997

Differential effects of neuroactive steroids on somatostatin and dopamine secretion from primary hypothalamic cell cultures.

This study investigated the effects of neuroactive steroids, which have been reported to modulate GABA-ergic transmission, on the secretion of somatostatin (SRIH) and also dopamine (DA) from primary rat hypothalamic cell cultures, where the release of both substances is regulated by a GABAA receptor-mediated inhibitory tone. Pregnenolone sulphate (PS), a negative allosteric modulator at the GABAA receptor, enhanced SRIH secretion in a time and dose-dependent manner (10(-12)-10(-8) M). This effect was reversed by muscimol (10(-8) M) and enhanced by bicuculline (10(-6) M), thus supporting an action of PS at the GABAA receptor. The release of endogenously synthesized dopamine (DA) was, however, unaffected by PS. A number of other steroids were also tested for their potential actions on SRIH and DA secretion. Allopregnanolone had slight but significant stimulatory actions on SRIH secretion, whereas tetrahydro-deoxycorticosterone (TH-DOC) markedly stimulated SRIH secretion with a bell-shaped dose response curve resembling that found for PS. The release of DA was unaffected by these neuroactive steroids but, unlike SRIH, DA release was stimulated by dehydroepiandrosterone sulphate (DHEAS). The results support the view that neuroactive steroids may play an important role in regulating some aspects of neuroendocrine function and they also provide the first demonstration of differential activities of neuroactive steroids within the hypothalamus at low, physiologically relevant concentrations. The results also raise the possibility that certain hypothalamic neuronal populations may possess uniquely different GABAA receptors and that such mechanisms may contribute to the functional development of the neuroendocrine system.

Animals↗
PubMed2006

Caveolins and the regulation of endothelial nitric oxide synthase in the heart.

Virtually all cell types within the myocardium express caveolae, where cell-specific isoforms of caveolin both maintain the structural organisation of these cholesterol-rich of the plasmalemma and serve as scaffolds for the dynamic constitution of "signalosomes", or hubs concentrating numerous transmembrane signaling proteins and their effectors. Analysis of the phenotype of mice with genetic deletion or overexpression of specific caveolin isoforms has provided key evidence for the importance of caveolins and caveolae in several aspects of the cardiovascular biology, including vascular contractility, lipid metabolism, angiogenesis, or the control of cardiac hypertrophy. Among specific protein-protein interactions involving caveolins in cardiac tissue, these genetic models unequivocally confirmed the functional importance of the dynamic association of the endothelial isoform of nitric oxide synthase (eNOS) for its post-translational regulation in endothelial cells and cardiac myocytes, which bears on the enzyme's capacity to modulate nitric oxide (NO)-dependent endothelial function, angiogenesis, and excitation-contraction coupling. We will review the current understanding of this regulation of eNOS (and potentially other NOS isoforms) through protein-protein interactions involving several G-protein-coupled receptors and other allosteric modulators in the context of emerging paradigms on the regulation of cardiac function by NO.

Animals↗
PubMed2000

Effect of AMPA receptor modulators on hippocampal and cortical function.

Attention has focused on drugs that modulate AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid) receptors because of their potential for enhancing memory and treating certain pathologies that involve glutamatergic neurotransmission. The aim of this study was to compare and contrast the functionality of positive allosteric modulators of AMPA receptors in the hippocampus and medial prefrontal cortex. Electrically stimulated EPSPs (excitatory postsynaptic potential) in the hippocampus were augmented by CX516 [(1-quinoxaline-6-ylcarbonyl)piperidine], aniracetam and 1-BCP [(1-(1,3-benzodioxol-5-ylcarbonyl)piperidine] and not by cyclothiazide. Using grease gap electrophysiology, it was found that the mode of application dramatically altered the effect of the modulators of AMPA-induced depolarization. When added simultaneously with AMPA, aniracetam, 1-BCP and CX516 augmented the response in the frontal cortex. However, in the hippocampus, only aniracetam and cyclothiazide augmented the response when simultaneously added to AMPA. Therefore, in addition to regional variations, there appears to be differences in modulator response dependent upon whether a response is generated endogenously or exogenously by AMPA.

Action Potentials↗
PubMed2000

Protein-protein interactions controlling nitric oxide synthases.

Nitric oxide (NO) biosynthesis is tightly regulated by a variety of mechanisms ranging from transcriptional to post-translational controls. Calmodulin has long been known to be an allosteric modulator of the three major NO synthases (NOS). Recent studies indicate that other proteins directly associate with NOS isoforms and regulate their activity or spatial distribution in the cell. Several proteins residing in or recruited to plasmalemmal caveolae of endothelial cells serve as allosteric regulators of endothelial NOS (eNOS). Caveolins, the resident scaffolding proteins of caveolae, and calmodulin undergo reciprocal Ca2+-dependent association and dissociation with eNOS in the caveolar membrane that inhibits (caveolins) and activates (calmodulin) eNOS activity. Other caveolar proteins appear to contribute to the eNOS-membrane complex, including the bradykinin B2 receptor, the angiotensin AT1 receptor, the CAT1 arginine transporter, and Hsp90. Direct interactions of a variety of proteins bearing PDZ domains with the PDZ domain of neuronal NOS (nNOS) have been shown to influence the subcellular distribution and/or activity of the enzyme in brain and muscle. One of these proteins, PSD-93, co-localizes with a subpopulation of nNOS in the macula densa. Although considerable emphasis has been placed on transcription as the principal step of regulation for inducible NOS (iNOS), our laboratory has recently defined a regulatory interaction of iNOS with Rho family GTPases. While the role of protein-eNOS interactions in the control of vascular tone has been increasingly clarified, the interactions and regulatory importance of protein association with nNOS and iNOS in the vasculature and kidney remains to be explored.

Animals↗
PubMed2003

Identification of a site in GluR1 and GluR2 that is important for modulation of deactivation and desensitization.

The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid subtype of ionotropic glutamate receptors consists of rapidly gating ion channels. Positive modulation of channel gating may slow gating kinetics through at least two distinct mechanisms, evidenced by the predominant slowing of either the rate of receptor desensitization or the rate of offset after agonist withdrawal (deactivation). This study compares the actions of two positive allosteric modulators [cyclothiazide, which modulates desensitization, and 1-(1,4-benzodioxan-6-ylcarbonyl)piperidine (CX546), which modulates deactivation] in a mutant shown previously to impede modulation by cyclothiazide. These experiments test the hypothesis that the point mutation, GluR1(S493T), would also cause a loss of modulation by CX546. Wild-type GluR1 through -4 receptors were modulated by CX546, as assayed by the potentiation of steady-state currents in the Xenopus laevis oocyte expression system. CX546 potentiated steady-state currents of both splice isoforms of GluR1. Modulation by CX546 was completely abolished in GluR1(S493T) and its homolog, GluR2(S497T), although this mutation did not affect apparent agonist affinity in the absence of CX546. Thus, the GluR1(S493T) mutation has a similar impairment of modulation by either cyclothiazide or CX546, indicating that some residues at the subunit interface of glutamate receptors play an important role in channel deactivation and desensitization.

Amino Acid Substitution↗
PubMed2005

N-terminal-mediated homomultimerization of prestin, the outer hair cell motor protein.

The outer hair cell lateral membrane motor, prestin, drives the cell's mechanical response that underpins mammalian cochlear amplification. Little is known about the protein's structure-function relations. Here we provide evidence that prestin is a 10-transmembrane domain protein whose membrane topology differs from that of previous models. We also present evidence that both intracellular termini of prestin are required for normal voltage sensing, with short truncations of either terminal resulting in absent or modified activity despite quantitative findings of normal membrane targeting. Finally, we show with fluorescence resonance energy transfer that prestin-prestin interactions are dependent on an intact N-terminus, suggesting that this terminus is important for homo-oligomerization of prestin. These domains, which we have perturbed, likely contribute to allosteric modulation of prestin via interactions among prestin molecules or possibly between prestin and other proteins, as well.

Allosteric Site↗
PubMed1999

Postnatal ethanol exposure blunts upregulation of GABAA receptor currents in Purkinje neurons.

Recently, we found that early postnatal ethanol exposure inhibits the maturation of GABAA receptors (GABAARs) in developing medial septum/diagonal band (MS/DB) neurons, suggesting that these receptors may represent a target for ethanol related to fetal alcohol syndrome (FAS). To determine whether GABAARs on other neurons are also sensitive to a postnatal ethanol insult, postnatal day (PD) 4-9, rat pups were artificially reared and exposed to ethanol (4.5 g kg-1 day-1, 10.2% v/v). The pharmacological profile of acutely dissociated cerebellar Purkinje cell GABAARs from untreated, artificially reared controls and ethanol-treated animals was examined with conventional whole-cell patch clamp recordings during PD 12-16 (juveniles) and PD 25-35 (young adults). For untreated animals, GABA (0.3-100 microM) consistently induced inward Cl- currents in a concentration-dependent manner showing an age-related increase in maximum response without change in EC50 or slope value. Acute ethanol (100 mM) consistently inhibited 3 microM GABA currents (10-20%); positive modulators, pentobarbital (10 microM), midazolam (1 microM) and loreclezole (10 microM), consistently potentiated; the negative modulator, Zn2+ (30 microM), inhibited GABA currents across both juvenile and young adult groups. Loreclezole potentiation increased while Zn2+ inhibition decreased with age in untreated Purkinje neurons. Postnatal ethanol exposure (PD 4-9) decreased GABAAR maximum current density in young adult Purkinje cells but not in juvenile neurons. However, sensitivity to allosteric modulators did not change after ethanol. These data are consistent with the hypothesis that postnatal ethanol exposure during the brain growth spurt can disturb GABAAR development across the brain, although the mechanism(s) underlying this action remains to be determined.

Allosteric Regulation↗
PubMed1992

High-affinity dextromethorphan and (+)-3-(-3-hydroxyphenyl)-N-(1-propyl)piperidine binding sites in rat brain. Allosteric effects of ropizine.

Dextromethorphan (DM) binds to high- and low-affinity sites in the rat brain. The high-affinity DM binding is inhibited by nonnarcotic antitussives, opipramol and sigma ligands with nanomolar affinities. Computer-assisted modeling of homologous and heterologous competition studies between DM and (+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine [(+)-3-PPP] were performed at pH 8.4. These experiments confirmed the existence of the common high-affinity DM1/sigma 1 site (R1) for which DM and (+)-3-PPP have Kd values of 20 and 10 nM, respectively. DM also binds to a second high-affinity site (R2, Kd, 20 nM) for which (+)-3-PPP has only micromolar affinity. Similarly, (+)-3-PPP binds to another high-affinity site (R3, Kd, 60 nM) for which DM has micromolar affinity. The common high-affinity DM1/sigma 1 site is allosterically modulated by the anticonvulsant ropizine, and is (+)-pentazocine sensitive, as is the homologous site in the guinea pig. However, in the rat the common DM1/sigma 1 site is 10 times smaller than in the guinea pig. This explains the apparently different effects of the allosteric modifiers in both species. The multiplicity of binding sites for DM and (+)-3-PPP resolved in this investigation will help to establish the physiological role and the pharmacological potential of the different sites. Meanwhile, the pharmacological effects of DM and sigma ligands cannot be summarily attributed to any particular binding site or receptor. This investigation also demonstrates that the use of multiple labeled and unlabeled ligands, combined with computer-assisted modeling, is essential to resolve multiple binding sites with similar affinities and to characterize the complex effects of allosteric modifiers.

Allosteric Regulation↗
PubMed2004

Bisquaternary caracurine V and iso-caracurine V salts as ligands for the muscle type of nicotinic acetylcholine receptors: SAR and QSAR studies.

The binding constants (K(i) values) of 24 caracurine V and 6 iso-caracurine V analogues for the muscle type of nicotinic ACh receptors (nAChR) from Torpedo californica were determined in a binding assay using (+/-)-[(3)H]epibatidine as a radioligand. The allyl alcohol group present in the iso-caracurine V ring system was found to be essential for high binding affinity. The most potent compounds are the dimethyl and di-(4-nitrobenzyl)-iso-caracurinium V salts 29 (18 nM), and 31 (79 nM), respectively. Compound 29 and the corresponding diallyl analogue 30 (350 nM) exhibited similar binding affinities as the equally substituted neuromuscular-blocking agents toxiferine I (14 nM) and alcuronium (234 nM), respectively. The SAR results were confirmed by QSAR studies, which additionally revealed that the presence of hydrogen-bond acceptor groups close to the quaternary nitrogen, is detrimental for the nicotinic binding affinity. The diallyl- and dimethylcaracurinium V salts 13 and 27, respectively, which are known to be among the most potent allosteric modulators of M(2) receptors (EC(50)=10 and 8nM, respectively), exhibited rather low nicotinic binding affinities for muscle type nAChR (K(i)=1.5 and 5.2 microM, respectively). Such a large difference in affinity suggests that it is possible to develop compounds with high muscarinic allosteric potency and low or negligible affinities for (alpha1)(2)beta1gammadelta nAChR. Additionally, the iso-caracurine V analogues with binding affinities comparable to those of (+)-tubocurarine and alcuronium could become a new class of neuromuscular-blocking agents.

Alkaloids↗
PubMed2003

Discriminative stimulus effects of positive GABAA modulators and other anxiolytics, sedatives, and anticonvulsants in untreated and diazepam-treated monkeys.

Positive GABAA modulators and other sedatives, anxiolytics, and anticonvulsants were used to evaluate mechanisms underlying the discriminative stimulus effects of midazolam in untreated monkeys and of flumazenil in monkeys treated with diazepam (5.6 mg/kg/day). Positive GABAA modulators at benzodiazepine (e.g., flunitrazepam and abecarnil) and neuroactive steroid sites (e.g., androsterone) substituted for midazolam in all monkeys; the neuroactive steroids dihydroandrosterone and epipregnanolone substituted for midazolam in two of three monkeys. All positive GABAA modulators attenuated flumazenil in diazepam-treated monkeys; doses of flunitrazepam and abecarnil larger than doses substituting for midazolam were required to attenuate flumazenil, whereas doses of neuroactive steroids smaller than doses substituting for midazolam attenuated flumazenil. Drugs with mechanisms that do not predominantly involve allosteric modulation of GABA (e.g., buspirone, ketamine, valproic acid, and diphenhydramine) did not substitute for midazolam or flumazenil. However, valproic acid enhanced the midazolam discriminative stimulus and attenuated the flumazenil discriminative stimulus; diphenhydramine attenuated the midazolam discriminative stimulus. These results suggest that drugs not sharing a mechanism of action with benzodiazepines can modulate the behavioral effects of benzodiazepines. In addition, this study demonstrates that endogenous ligands, presumably by acting at neuroactive steroid sites on the GABAA receptor complex, share discriminative stimulus effects with benzodiazepines. This study also suggests that positive GABAA-modulating neuroactive steroids are especially potent in attenuating behavioral effects that are related to diazepam withdrawal.

Animals↗
PubMed2002

Cholesterol and steroid hormones: modulators of oxytocin receptor function.

The function and physiological regulation of the oxytocin-receptor system is strongly steroid-dependent. This is, unexpectedly, only partially reflected by the promoter sequences in the oxytocin receptor and favors the idea that posttranscriptional mechanisms may also play a significant role for the physiological regulation of the oxytocin-receptor system. Our data indicate that cholesterol acts as an allosteric modulator of the oxytocin receptor and stabilizes both membrane-associated and solubilized OT receptors in a high-affinity state for agonists and antagonists. Moreover, high-affinity OT receptors are 2-fold enriched in cholesterol-rich plasma membrane domains in HEK293 fibroblasts stably expressing the human OT receptor. Biochemical data suggest a direct and cooperative molecular interaction of cholesterol molecules with OT receptors. To localize the cholesterol interacting domain of the oxytocin receptor the C-terminal part including the last two transmembrane domains have been exchanged by the corresponding sequences of the cholecystokinin type B receptor, which is functionally not dependent on cholesterol. Concerning its ligand-binding behavior this chimeric receptor protein showed the same dependence on cholesterol and its analogues as the wild type oxytocin receptor. From mutagenesis experiments and studies with receptor chimera between the OTR and cholecystokinin type B receptor, we conclude that a major part of the cholesterol interacting domain may be localized in the first part of the oxytocin receptor, possibly in a domain nearby the agonist binding site. Progesterone is considered to be essential to maintain the uterine quiescence. High concentrations of progesterone (> 10 microM) attenuate or block the signaling of several GPCRs, including the OT receptor via a fast, reversible and non-genomic pathway. Progesterone is known to inhibit both cholesterol biosynthesis and the intracellular trafficking of cholesterol. We therefore test the hypothesis that progesterone affects the signal transduction and subdomain localization of receptors via its influence on cholesterol trafficking. Since cholesterol-rich subdomains (rafts) are considered to be organization centers for cellular signal transduction, changes of the level or distribution of cholesterol may have profound effects on receptor-mediated signaling in general. Using fluorescence recovery after photobleaching (FRAP) measurements with GFP-tagged oxytocin receptors the influence of steroids on the mobility and distribution of the oxytocin receptor in the plasma membrane was analyzed. Progesterone had no effect on the lateral mobility of the oxytocin receptor, but it led to marked inhibition of cellular motility such as vesicle trafficking and movements of filopodia. Non-genomic effects of progesterone and estradiol with respect to receptor signaling as well as the influence of cholesterol on signal transduction will be discussed in more detail.

Allosteric Regulation↗
PubMed1987

Ca2+-activated ryanodine binding: mechanisms of sensitivity and intensity modulation by Mg2+, caffeine, and adenine nucleotides.

The Ca2+-ryanodine receptor complex is a functional unit at the terminal cisternae (TC) of the sarcoplasmic reticulum (SR) whose proteins comprise the Ca2+ release channels which may be involved in excitation-contraction coupling. Ca2+, Mg2+, caffeine, and adenine nucleotides, but not inositol 1,4,5-trisphosphate, may exert their inotropic effects on skeletal muscle SR by direct allosteric modulation of the [3H]ryanodine-binding site. Micromolar Ca2+ is primarily responsible for activating [3H]ryanodine binding by regulating receptor site density, affinity, and cooperativity. Mg2+ reduces the sensitivity to Ca2+ activation by directly competing with Ca2+ for the activator site. However, inhibition by Mg2+ is overcome in the presence of beta,gamma-methyleneadenosine 5'-triphosphate (AMP-PCP; 1 mM) or caffeine (20 mM). Caffeine dramatically increases the affinity of the Ca2+ activator site for Ca2+, whereas AMP-PCP or cAMP enhances the gating efficiency or the lifetime of the open state of the TC SR channel. A kinetic model is proposed for four functional domains of the Ca2+-ryanodine receptor complex: the Ca2+-regulatory domain which binds Ca2+ with microM affinity is primarily responsible for gating the Ca2+ channel of the TC SR in a cooperative manner, and is inhibited by mM Mg2+ by direct competition for the activator site which appears to contain critical sulfhydryl groups; a Ca2+-activate alkaloid binding domain in close proximity to the channel which binds ryanodine with nM affinity and rapidly occludes upon complex formation; a domain which binds caffeine with low (greater than mM) affinity and directly influences the sensitivity of the Ca2+-regulatory site; and a domain which binds adenine nucleotides with intermediate affinity (less than mM), does not require phosphorylation, and intensifies the Ca2+ signal which triggers opening of the Ca2+-release channel.

Adenine Nucleotides↗
PubMed2005

G protein-coupled receptors: a count of 1001 conformations.

G protein-coupled receptors (GPCRs) were initially regarded to adopt an inactive and an active conformation and to activate a single type of G protein. Studies with recombinant cell systems have led to a more complex picture. First, GPCRs can activate distinct G protein species. Second, GPCR multistate models have been invoked to explain their complex behaviour in the presence of agonists, antagonists and other binding partners. The occurrence of intermediate receptor conformational states during GPCR activation and antagonist binding is suggested by fluorescence measurements and studies with constitutively active receptor mutants and insurmountable antagonists. Different agonists may trigger distinct effector pathways through a single receptor by dictating its preference for certain G proteins (i.e. 'agonist trafficking'). Structural modification and exogenous and endogenous (e.g. other cellular proteins, lipids) allosteric modulators also affect ligand-GPCR interaction and receptor activation. These new developments in GPCR research could lead to the development of more selective therapeutic drugs.

Allosteric Regulation↗
PubMed2001

Modulation of native and recombinant GABA(A) receptors by endogenous and synthetic neuroactive steroids.

Upon administration, certain pregnane steroids produce clear behavioural effects including, anxiolysis, sedation, analgesia, anaesthesia and are anti-convulsant. This behavioural profile is characteristic of compounds that act to enhance the actions of GABA acting at the GABA(A) receptor. In agreement, numerous studies have now demonstrated these steroids to be potent, positive allosteric modulators of the GABA(A) receptor. The pregnane steroids are synthesized in the periphery by endocrine glands such as the adrenals and the ovaries, but are also made by neurons and glial cells in the central nervous system itself. Hence, these compounds could play both an endocrine and a paracrine role to influence neuronal excitability by promoting inhibition. Here we review evidence that the pregnane steroids are highly selective and extremely potent GABA(A) receptor modulators and that their effects at 'physiological' concentrations (low nanomolar) may be influenced by the subunit composition of the GABA(A) receptor. This feature may underlie recent findings demonstrating the effects of the neurosteroids on inhibitory synaptic transmission to be brain region dependent, although recent reports suggest that phosphorylation mechanisms may additionally influence neurosteroid sensitivity of the GABA(A) receptor. Numerous synthetic steroids have been synthesized in an attempt to therapeutically exploit the behavioural effects of the pregnane steroids and progress with this approach will be discussed. However, the demonstration that the steroids may be made within the central nervous system offers the alternative strategy of targeting the enzymes that synthesize/metabolise the neurosteroids to exploit this novel endocrine/paracrine interaction.

Animals↗