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PubMed2004

Fishing for allosteric sites on GABA(A) receptors.

GABA(A) receptors have structural and functional homology with a super-family of cys-loop ligand-gated ion channel receptors including the nicotinic acetylcholine receptors. Amino acid residues involved in ligand-binding pockets are homologous among super-family members, leading to the multiple-loop model of binding sites situated at subunit interfaces, validated by structural studies on the nicotinic acetylcholine receptor and water-soluble snail acetylcholine binding protein. This article will briefly review the literature on the agonist binding sites on the receptor super-family, and then describe the current situation for attempts to identify sites for allosteric modulators on the GABA(A) receptors. A combination of mutagenesis and photoaffinity labeling with anesthetic ligands has given some leads in this endeavor. Current work by others and ourselves focuses on three putative sites for modulators: (1) within the ion channel domain TM2, near the extracellular end; (2) the agonist binding sites and homologous pockets at other subunit interfaces of the pentameric receptor; and (3) on the linker region stretching from the agonist site loop C to the top of the TM1 region. It is likely that concrete structural information will be forthcoming soon.

Allosteric Site↗
PubMed1999

Dual control of cytochrome-c oxidase activity by female sex steroids.

Female sex steroids modify cytochrome-c oxidase (COX) activity in brown adipose tissue. To check the possibility of extending this modulating effect upon oxidative capacity to other tissues, COX activity was measured in different tissues from cold-acclimated female rats that were (1) intact in proestrus and diestrus I, (2) ovariectomized or (3) ovariectomized and treated with oestradiol and/or progesterone. In intact rats, COX activity varied within the oestrous cycle in brown adipose tissue and soleus muscle. Ovariectomy induced an increase in COX activity in most of the tissues studied, an increase reversed only after 10 days of treatment with oestradiol and/or progesterone. These results indicate both a short-term (oestrous cycle) and a long-term (ovariectomy) control of COX activity by female sex steroids, probably mediated by allosteric modulation and control of the enzyme synthesis respectively. In thermogenic tissues, that is brown adipose tissue and skeletal muscles, the short-term control is interpreted as a cooperation between tissues to fulfil the requirements of temperature maintenance.

Acclimatization↗
PubMed2005

Muscarinic allosteric enhancers of ligand binding: pivotal pharmacophoric elements in hexamethonio-type agents.

Bisphthalimidopropyl-substituted hexamethonio compounds have been established as allosteric modulators of ligand binding to muscarinic acetylcholine receptors. Enhancers of ligand binding are of special interest. This study aimed to unravel the structural elements inducing positive cooperativity with the binding of an antagonist. [(3)H]-N-methylscopolamine binding to muscarinic M(2) receptors was measured in porcine heart homogenates. Dimethylation, but not monomethylation, of the lateral propyl chain in combination with an affinity increasing aromatic imide moiety, such as a 5-methylphthalimide and naphthalimide, on the same side of the molecule shifts the cooperativity toward positive values, resulting in enhancers of antagonist binding. Thus, lateral side chain dimethylation is a pivotal pharmacophoric element for positive cooperativity in hexamethonio-type muscarinic allosteric agents.

Allosteric Regulation↗
PubMed1999

Using a radioalloster to test predictions of the cooperativity model for gallamine binding to the allosteric site of muscarinic acetylcholine M(2) receptors.

The muscarinic M(2) receptor contains an orthosteric and an allosteric site. Binding of an allosteric agent may induce a shift alpha of the equilibrium dissociation constant K(D) of a radioligand for the orthosteric site. According to the cooperativity model, the K(A) of alloster binding is expected to be shifted to an identical extent depending on whether the orthosteric site is occupied by the orthoster or not. Here, the novel radioalloster [(3)H]dimethyl-W84 (N,N'-bis[3-(1,3-dihydro-1, 3-dioxo-4-methyl-2H-isoindol-2-yl)propyl]-N,N,N',N'-tetramethyl-1, 6-hexanediaminium diiodide) was applied to directly measure the K(A) shift induced for the prototype allosteric modulator gallamine by binding of N-methylscopolamine (NMS) to the orthosteric site of porcine heart M(2) receptors (4 mM Na(2)HPO(4), 1 mM KH(2)PO(4), pH 7.4; 23 degrees C; data are means +/- S.E.). First, in the common way, the concentration-dependent inhibition by gallamine of [(3)H]NMS equilibrium binding was measured and analyzed using the cooperativity model, which yielded for the affinity of gallamine binding at free receptors a pK(A)= 8.35 +/- 0.09 and a cooperativity factor alpha = 46 (n = 5). The dissociation constant for gallamine binding at NMS-occupied receptors was predicted as p(alpha. K(A)) = 6.69. Labeling of the allosteric site by [(3)H]dimethyl-W84 allowed the measure of competitive displacement curves for gallamine. The K(i) for gallamine at free receptors amounted to pK(i,-NMS) = 8.27 +/- 0.39 (n = 5), which is in line with the prediction of the cooperativtiy model. In the presence of 1 microM NMS, to occupy the orthosteric site, gallamine displaced [(3)H]dimethyl-W84 with pK(i, +NMS) = 6.60 +/- 0.19 (n = 3). Thus, the NMS-induced pK(i) shift amounted to 47, which matches the predicted value of alpha = 46. These results validate the cooperativity model.

Allosteric Site↗
PubMed1993

Sigma receptors modulate nicotinic receptor function in adrenal chromaffin cells.

Neither the physiological function of sigma (sigma) receptors nor the cellular mechanism responsible for the pharmacological effects of sigma receptor ligands is known. We now report that sigma receptor ligands noncompetitively inhibit nicotine-stimulated catecholamine release from bovine adrenal chromaffin cells in a concentration-dependent and reversible manner. The rank order of potency of ligands to inhibit nicotine-stimulated catecholamine release is significantly correlated (P < 0.005) with that observed in radioligand binding assays selective for the sigma 1 receptor subtype. This naltrexone-insensitive effect is paralleled by an inhibition of nicotine-stimulated increases in [Ca2+]i. Sigma ligands were without effect on catecholamine release or [Ca2+]i in the absence of nicotine. In addition, nicotine accelerated the association of the sigma receptor selective radioligand, [3H](+)pentazocine, to adrenal medullary homogenates while having no effect on the rate of ligand dissociation, consistent with a sigma ligand binding site closely associated with and allosterically modulated by the nicotinic acetylcholine receptor. Thus, the actions of agonists at the nicotinic acetylcholine receptor in bovine chromaffin cells are modulated by sigma 1 receptor selective ligands.

Adrenal Medulla↗
PubMed1996

Progesterone induces changes in sleep comparable to those of agonistic GABAA receptor modulators.

There is much evidence that progesterone has hypnotic anesthetic properties. In this vehicle-controlled study, we examined the effects of three doses of progesterone (30, 90, and 180 mg/kg) administered intraperitoneally at light onset on sleep in rats. Progesterone dose dependently shortened non-rapid eye movement sleep (NREMS) latency, lengthened rapid eye movement sleep (REMS) latency, decreased the amount of wakefulness and REMS, and markedly increased pre-REMS, an intermediate state between NREMS and REMS. Progesterone also elicited dose-related changes in sleep state-specific electroencephalogram (EEG) power densities. Within NREMS, EEG activity was reduced in the lower frequencies (< or = 7 Hz) and was enhanced in the higher frequencies. Within REMS, EEG activity was markedly enhanced in the higher frequencies. The effects were maximal during the first postinjection hours. The concentrations of progesterone and the progesterone metabolites 3 alpha-hydroxy-5 alpha-pregnan-20-one and 3 alpha-hydroxy-5 beta-pregnan-20-one, both positive allosteric modulators of gamma-aminobutyric acid A (GABAA) receptors, were determined at different time intervals after vehicle and 30 or 90 mg/kg progesterone. Progesterone administration resulted in dose-dependent initially supraphysiological elevations of progesterone and its metabolites in the plasma and brain, which were most prominent during the first hour postinjection. The effects of progesterone on sleep closely resemble those of agonistic modulators of GABAA receptors such as benzodiazepines and correlate well with the increases in the levels of its GABAA agonistic metabolites. These observations suggest that the hypnotic effects of progesterone are mediated by the facilitating action of its neuroactive metabolites on GABAA receptor functioning.

Animals↗
PubMed2006

Mutations of L293 in transmembrane two of the mouse 5-hydroxytryptamine3A receptor alter gating and alcohol modulatory actions.

1 The goal of this study was to determine whether mutations of L293 at the 15' position of TM2 in the 5-HT(3A) receptor alter macroscopic current kinetics, and if these changes could account for alterations in alcohol modulation. Receptor function was assessed in Xenopus oocytes under voltage-clamp and in HEK293 cells with whole-cell patch-clamp recording and rapid drug application. 2 Examination of responses of L293C and L293S receptors to agonist alone revealed enhanced activation, deactivation, and desensitization rates relative to the wild-type receptor. The L293G mutation produced marked slowing of deactivation and desensitization rates. Increased potency of 5-HT and increased efficacy of the partial agonist, DA, was also observed in these mutant receptors. 3 Ethanol and trichloroethanol (TCEt) enhancement of receptor function was reduced or eliminated in receptors containing L293 mutations to C, G, or S. The L293I mutant receptor retained ethanol and TCEt sensitivity. Ethanol and TCEt enhanced activation rate in the wild-type, but not the L293G and L293S receptors. No relationship was observed between any physicochemical property of the substituted amino acids and the change in alcohol potentiation of function. 4 The changes in receptor-channel properties in the mutant receptors support the idea that the L293 residue has important roles in channel gating. Our findings indicate that loss of allosteric modulation by alcohols is not related in any simple way to changes in channel kinetic properties brought about by L293 mutants. We did not observe any evidence that L293 is part of an alcohol binding site.

Allosteric Regulation↗
PubMed2001

An NMDA receptor signaling complex with protein phosphatase 2A.

Regulation of protein phosphatase 2A (PP2A) activity and NMDA receptor (NMDAR) phosphorylation state contribute to the modulation of synaptic plasticity, yet these two mechanisms have not been functionally linked. The NMDAR subunit NR3A is equipped with a unique carboxyl domain that is different from other NMDAR subunits. We hypothesized that the NR3A C-terminal intracellular domain might serve as synaptic anchor for the phosphatase in the developing CNS. A cDNA library was screened by the yeast two-hybrid method using the NR3A carboxyl domain as the bait. The catalytic subunit of the serine-threonine PP2A was found to be associated with the NR3A carboxyl domain. Immunoprecipitation studies indicated that the NR3A subunit formed a stable complex with PP2A in the rat brain in vivo. Association of PP2A with NMDARs led to an increase in the phosphatase activity of PP2A and the dephosphorylation of serine 897 of the NMDAR subunit NR1. Stimulation of NMDARs led to the dissociation of PP2A from the complex and the reduction of PP2A activity. A peptide corresponding to the PP2A-NR3A binding domain functioned as a negative regulator of PP2A activity. These data suggest that NMDARs are allosteric modulators of PP2A, which in turn controls their phosphorylation state. The data delineate a mechanistic model of the dynamic regulation of a PP2A-NMDAR signaling complex, mediated by the interaction of NR3A and PP2A, and suggest a novel NMDAR-mediated signaling mechanism in addition to the traditional ionotropic functions of NMDARs.

Allosteric Regulation↗
PubMed2000

Functional coupling of oxygen binding and vasoactivity in S-nitrosohemoglobin.

S-Nitrosohemoglobin (SNO-Hb) is a vasodilator whose activity is allosterically modulated by oxygen ("thermodyamic linkage"). Blood vessel contractions are favored in the oxygenated structure, and vasorelaxant activity is "linked" to deoxygenation, as illustrated herein. We further show that transnitrosation reactions between SNO-Hb and ambient thiols transduce the NO-related bioactivity, whereas NO itself is inactive. One remaining problem is that the amounts of SNO-Hb present in vivo are so large as to be incompatible with life were all the S-nitrosothiols transformed into bioactive equivalents during each arterial-venous cycle. Experiments were therefore undertaken to address how SNO-Hb conserves its NO-related activity. Our studies show that 1) increased O(2) affinity of SNO-Hb (which otherwise retains allosteric responsivity) restricts the hypoxia-induced allosteric transition that exchanges NO groups with ambient thiols for vasorelaxation; 2) some NO groups released from Cys(beta93) upon transition to T structure are autocaptured by the hemes, even in the presence of glutathione; and 3) an O(2)-dependent equilibrium between SNO-Hb and iron nitrosylhemoglobin acts to conserve NO. Thus, by sequestering a significant fraction of NO liberated upon transition to T structure, Hb can conserve NO groups that would otherwise be released in an untimely or deleterious manner.

Allosteric Regulation↗
PubMed2002

Enhancement of neuronal nicotinic receptor activity of rat chromaffin cells by a novel class of peptides.

The N-terminal 1-7 fragment of the neuropeptide CGRP inhibits neuronal nicotinic acetylcholine receptors (nAChRs) of rat chromaffin cells. To identify the structural motif responsible for this action, we investigated the effects of shorter CGRP fragments on patch-clamped rat chromaffin cells in culture. CGRP(1-6) evoked no direct change in baseline current or input conductance, but it strongly potentiated inward currents induced by very fast, nondesensitizing applications of nicotine. Potentiation was use independent and present even when coapplied with nicotine. The action of CGRP(1-6) was voltage independent and agonist independent. Because equimolar concentrations of CGRP(1-6) and CGRP(1-7) left nicotine-induced submaximal currents unchanged, these peptides presumably acted via a similar site through which they generated opposite effects. This observation also suggests that a single amino acid deletion could transform a peptide antagonist into a potentiating one. Deleting one amino acid from the COO(-) end of the CGRP(1-6) sequence yielded CGRP(1-5), which retained smaller potentiating activity. Even the CGRP(1-4) fragment possessed slight potentiation, which was lost with CGRP(1-3). CGRP(1-6) preferentially potentiated small over large responses to nicotine. One possibility is that CGRP(1-6) interacted with nAChRs like an allosteric modulator (e.g., physostigmine). Coapplication of enhancing concentrations of physostigmine and CGRP(1-6) led to linear summation of the individual effects, while CGRP(1-6) could partly reverse the depression by a large concentration of physostigmine. These data indicate functionally distinct sites of action for CGRP(1-6) and physostigmine. Potentiation of nicotinic receptors by CGRP(1-6) and its derivatives suggests them to be a new class of molecules enhancing activity mediated by nAChRs.

Allosteric Site↗
PubMed2004

Pentobarbital differentially modulates alpha1beta3delta and alpha1beta3gamma2L GABAA receptor currents.

GABAA receptors are modulated by a variety of compounds, including the neurosteroids and barbiturates. Although the effects of barbiturates on alphabetagamma isoforms, thought to dominate phasic (synaptic) GABAergic inhibition, have been extensively studied, the effects of pentobarbital on kinetic properties of alphabetadelta GABAA receptors, thought to mediate tonic (extra- or perisynaptic) inhibition, are unknown. Using ultrafast drug delivery and single channel recording techniques, we demonstrate isoform-specific pentobarbital modulation of low-efficacy, minimally desensitizing alpha1beta3 currents and high-efficacy, rapidly desensitizing alpha1beta3gamma2L currents. Specifically, with saturating concentrations of GABA, pentobarbital substantially potentiated peak alpha1beta3delta receptor currents but failed to potentiate peak alpha1beta3gamma2L receptor currents. Also, pentobarbital had opposite effects on the desensitization of alpha1beta3delta (increased) and alpha1beta3gamma2L (decreased) receptor currents evoked by saturating GABA. Pentobarbital increased steady-state alpha1beta3delta receptor single channel open duration primarily by introducing a longer duration open state, whereas for alpha1beta3gamma2L receptor channels, pentobarbital increased mean open duration by increasing the proportion and duration of the longest open state. The data support previous suggestions that GABA may be a partial agonist at alphabetadelta isoforms, which may render them particularly sensitive to allosteric modulation. The remarkable increase in gating efficacy of alpha1beta3delta receptors suggests that alphabetadelta isoforms, and by inference tonic forms of inhibition, may be important targets for barbiturates.

Cells, Cultured↗
PubMed2004

Abnormal benzodiazepine and zinc modulation of GABAA receptors in an acquired absence epilepsy model.

Brain cholesterol synthesis inhibition (CSI) at a young age in rats has been shown to be a faithful model of acquired absence epilepsy, a devastating condition for which few therapies or models exist. We employed the CSI model to study cellular mechanisms of acquired absence epilepsy in Long-Evans Hooded rats. Patch-clamp, whole-cell recordings were compared from neurons acutely dissociated from the nucleus reticularis of thalamus (nRt) treated and untreated with a cholesterol synthesis inhibitor, U18666A. In U18666A-treated animals, 91% of rats developed EEG spike-waves (SWs). Patchclamp results revealed that although there was no remarkable change in GABAA receptor affinity, both a loss of ability of benzodiazepines to enhance GABAA-receptor responses and an increase of Zn2+ inhibition of GABAA-receptor responses of nRt neurons occurred in Long-Evans Hooded rats previously administered U18666A. This change was specific, since no significant changes were found in neurons exposed to the GABA allosteric modulator, pentobarbital. Taken collectively, these findings provide evidence for abnormalities in benzodiazepine and Zn2+ modulation of GABAA receptors in the CSI model, and suggest that decreased gamma2 subunit expression may underlie important aspects of generation of thalamocortical SWs in atypical absence seizures. The present results are also consistent with recent findings that mutation of the gamma2 subunit of the GABAA receptor changes benzodiazepine modulation in families with generalized epilepsy syndromes.

Androstenes↗
PubMed1997

Modulation of the enzymatic properties of protein phosphatase 2A catalytic subunit by the recombinant 65-kDa regulatory subunit PR65alpha.

All protein phosphatase 2A (PP2A) holoenzymes contain a 36-kDa catalytic subunit (PP2Ac) and a regulatory subunit of 65 kDa (PR65). We have studied the interaction between PP2Ac and PR65 in an in vitro system, using PP2Ac isolated from rabbit skeletal muscle and recombinant PR65alpha expressed in bacteria or insect cells. Bacterially expressed PR65alpha exhibited identical biochemical properties to the protein expressed and isolated from the baculoviral expression system. The association of recombinant PR65 with PP2Ac was very tight (K(D)app = 85 pM) and led to a suppression of PP2A activity, which was maximal (70-80%) when phosphoproteins were used as substrates. When less-structured or smaller substrates (such as phosphopeptides) were used, this inhibition was only 30%. PR65 stimulated PP2Ac activity when the assays were performed in the presence of polycations. This indicates that the PR65 not only serves the previously predicted structural role as a molecular scaffold, but also allosterically modulates the enzymatic properties of PP2Ac. Furthermore, we identified a site of interaction between PP2Ac and PR65alpha by disruption of a stretch of basic amino acids by introduction of a glutamate at position 416. This produced an almost 100-fold reduced affinity for PP2Ac and indicated that this basic motif is an important determinant for the interaction of PR65 and PP2Ac.

Amino Acid Sequence↗
PubMed2005

The effect of dehydroepiandrosterone sulfate and allopregnanolone sulfate on the binding of [(3)H]ifenprodil to the N-methyl-d-aspartate receptor in rat frontal cortex membrane.

Neurosteroids have been shown to modulate the N-methyl-d-aspartate (NMDA) receptor function. Dehydroepiandrosterone sulfate (DHEAS) is shown to participate in memory and learning processes as well as preventing glutamate neurotoxicity in hippocampus. In this study we have focused on the modulatory effect of neurosteroids on ifenprodil binding to the NR2B subunit of the NMDA receptor. We show that DHEAS and allopregnanolone sulfate (ALLOPREGS) exert different effects on the [(3)H]ifenprodil binding at 10, 30 or 100 nM, corresponding to physiological concentrations. The effects include changes in the ifenprodil displacement curve, changing it from a one-site fit into a two-site fit leaving B(max), K(d) and K(off) unaffected. Our results indicate that DHEAS and ALLOPREGS induce an allosteric modulation of the NMDA receptor, an observation that might contribute to the understanding of the effects of these neurosteroids.

Animals↗
PubMed2004

A region in the seven-transmembrane domain of the human Ca2+ receptor critical for response to Ca2+.

Of 12 naturally occurring, activating mutations in the seven-transmembrane (7TM) domain of the human Ca2+ receptor (CaR) identified previously in subjects with autosomal dominant hypocalcemia (ADH), five appear at the junction of TM helices 6 and 7 between residue Ile819 and Glu837. After identifying a sixth activating mutation in this region, V836L, in an ADH patient, we studied the remaining residues in this region to determine whether they are potential sites for activating mutations. Alanine-scanning mutagenesis revealed five additional residues in this region that when substituted by alanine led to CaR activation. We also found that, whereas E837A did not activate the receptor, E837D and E837K mutations did. Thus, region Ile819-Glu837 of the 7TM domain represents a "hot spot" for naturally occurring, activating mutations of the receptor, and most of the residues in this region apparently maintain the 7TM domain in its inactive configuration. Unique among the residues in this region, Pro823, which is highly conserved in family 3 of the G protein-coupled receptors, when mutated to either alanine or glycine, despite good expression severely impaired CaR activation by Ca2+. Both the P823A mutation and NPS 2143, a negative allosteric modulator that acts on the 7TM through a critical interaction with Glu837, blocked activation of the CaR by various ADH mutations. These results suggest that the 7TM domain region Ile819-Glu837 plays a key role in CaR activation by Ca2+. The implications of our finding that NPS 2143 corrects the molecular defect of ADH mutations for treatment of this disease are also discussed.

Alanine↗
PubMed2001

Modulation by nucleotides of binding sites for [3H]glibenclamide in rat aorta and cardiac ventricular membranes.

Radioligand binding techniques were employed to determine the modulation by nucleotides of the specific [3H]glibenclamide (Gli) binding to rat aortic and cardiac ventricular preparations. Saturation analysis revealed a single binding site with K(D) value of 31.3 nM and Bmax of 180 fmol/mg wet weight in aortic preparations. We also observed that [3H]Gli bound reversibly and specifically to cardiac membranes. Unlabeled glibenclamide displaced [3H]Gli-specific binding of cardiac membranes completely with K(I) of 54.4 nM. In cardiac membranes, adenosine triphosphate (ATP), adenosine diphosphate (ADP), and uridine diphosphate (UDP) (from 0.01-5 mM) concentration dependently inhibited [3H]Gli binding independent of Mg2+. The values of K(I) were 0.47, 0.22, and 0.58 mM, respectively. However, in aortic preparations, [3H]Gli-specific binding was increased by ATP of 5 and 10 mM and showed a biphasic response to ADP. At concentrations to 1 mM, ADP inhibited binding; above 5 mM, the specific [3H]Gli binding was increased. UDP did not alter the binding up to 5 mM. In the presence of Mg2+ (20 mM), the inhibitory effects of ATP (0.01-1 mM) or ADP (0.01-5 mM) on the binding in cardiac membranes were abolished, whereas the facilitatory effects of ATP or ADP in aortic preparations were strengthened. Analysis of kinetics showed that the time of [3H]Gli association and dissociation in cardiac and aortic preparations was monophasic. The association was delayed with dissociation unchanged by ATP, ADP, and UDP of 1 mM, respectively, in cardiac membranes. In aorta, however, at the same concentration ATP accelerated association and retarded dissociation and vice versa for ADP. Association and dissociation were not changed by UDP of 5 mM. We conclude that ATP, ADP, and UDP are all major allosteric modulators of K(ATP) channels and they affect the antagonist binding to heart (sulfonylurea receptor 2A) and aorta (sulfonylurea receptor 2B) differently.

Adenosine Diphosphate↗
PubMed2003

ATP binding modulates the nucleic acid affinity of hepatitis C virus helicase.

The helicase of hepatitis C virus (HCV) unwinds nucleic acid using the energy of ATP hydrolysis. The ATPase cycle is believed to induce protein conformational changes to drive helicase translocation along the length of the nucleic acid. We have investigated the energetics of nucleic acid binding by HCV helicase to understand how the nucleotide ligation state of the helicase dictates the conformation of its nucleic acid binding site. Because most of the nucleotide ligation states of the helicase are transient due to rapid ATP hydrolysis, several compounds were analyzed to find an efficient unhydrolyzable ATP analog. We found that the beta-gamma methylene/amine analogs of ATP, ATPgammaS, or [AlF4]ADP were not effective in inhibiting the ATPase activity of HCV helicase. On the other hand, [BeF3]ADP was found to be a potent inhibitor of the ATPase activity, and it binds tightly to HCV helicase with a 1:1 stoichiometry. Equilibrium binding studies showed that HCV helicase binds single-stranded nucleic acid with a high affinity in the absence of ATP or in the presence of ADP. Upon binding to the ATP analog, a 100-fold reduction in affinity for ssDNA was observed. The reduction in affinity was also observed in duplex DNA with 3' single-stranded tail and in RNA but not in duplex DNA. The results of this study indicate that the nucleic acid binding site of HCV helicase is allosterically modulated by the ATPase reaction. The binding energy of ATP is used to bring HCV helicase out of a tightly bound state to facilitate translocation, whereas ATP hydrolysis and product release steps promote tight rebinding of the helicase to the nucleic acid. On the basis of these results we propose a Brownian motor model for unidirectional translocation of HCV helicase along the nucleic acid length.

Adenosine Triphosphate↗
PubMed1995

NMDA-mediated modulation of gamma-aminobutyric acid type A receptor function in cerebellar granule neurons.

GABAA receptors are ligand-gated CI- ion channels with multiple clinically relevant drug-recognition sites. We have previously shown that stimulation of N-methyl-D-aspartic acid (NMDA)-specific glutamate receptors quantitatively alters selected GABAA receptor subunit mRNAs and proteins in primary cultures of rat cerebellar granule neurons. We used whole-cell recordings of GABA-elicited CI- currents and flunitrazepam binding experiments in granule cell cultures maintained in low K+ (12.5 mM), cells maintained in low K+ and treated with a single dose of NMDA (10 microM), and cell cultures maintained in depolarizing concentrations of K+ (25 mM). The EC50 obtained from the dose-response curves for GABA in eliciting a maximal response was comparable in neurons maintained in high K+ or in low K+ and treated with a single dose of NMDA, but that it increased significantly in cells maintained in low K+. The potentiation of GABA-gated CI- currents by flunitrazepam increased significantly, while the negative allosteric modulator methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) was significantly more effective in cultures either maintained in high K+ or treated with NMDA. This was coincident with a twofold increase in the Bmax associated with flunitrazepam binding. To further characterize the receptor assemblies present in the depolarization and NMDA induced paradigms, the Zn(2+)-induced inhibition of GABA-gated CI- currents was reduced as was the inhibition mediated by furosemide. Our data indicate that GABAA receptor assemblies alter their composition in response to excitatory afferent receptor stimulation.

Animals↗