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PubMed2005

Type 10 17beta-hydroxysteroid dehydrogenase catalyzing the oxidation of steroid modulators of gamma-aminobutyric acid type A receptors.

The steroids allopregnanolone and allotetrahydrodeoxycorticosterone (3alpha,5alpha-THDOC) are positive allosteric modulators of GABA(A) receptors, generated by the reduction of 5alpha-dihydroprogesterone (5alpha-DHP) and 5alpha-DHDOC, respectively, under the catalysis of human type 3 3alpha-hydroxysteroid dehydrogenase (HSD). However, brain enzymes catalyzing the conversion of such tetrahydrosteroids back to the corresponding 5alpha-dihydrosteroids remain to be identified. Characterization of human type 10 17beta-HSD provides a new insight into its importance for the oxidation of steroid modulators of GABA(A) receptors. The apparent catalytic efficiency (k(cat)/K(m)) of this enzyme for the oxidation of allopregnanolone and 3alpha,5alpha-THDOC are 432 and 1381 min(-1) mM(-1), respectively. This enzyme has negligible 3-ketosteroid reductase activity for 5alpha-DHP and 5alpha-DHDOC even in an acidic environment. Immunoreactivity against 17beta-HSD10 was found in a number of neuronal populations. Taken together, evidence suggests that 17beta-HSD10 is the brain enzyme capable of catalyzing the oxidation of steroid modulators of GABA(A) receptors.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed2007

Modulation of P2X7 receptor functions by polymyxin B: crucial role of the hydrophobic tail of the antibiotic molecule.

BACKGROUND AND PURPOSE: P2X7 is a membrane receptor for extracellular ATP which is highly expressed in dendritic cells, macrophages and microglia where it mediates pro-inflammatory responses. The antibiotic polymyxin B, which binds to and neutralizes the toxic residue of bacterial lipopolysaccharide, greatly amplifies cellular responses mediated by the P2X7 receptor. However, the molecular mechanism involved is so far unknown. EXPERIMENTAL APPROACH: We investigated the effects of polymyxin B and polymyxin B nonapeptide (PMBN) which is the deacylated amino derivative of polymyxin B lacking the N-terminal fatty amino acid 6-methylheptanoic/octanoic-Dab residue, in human macrophages and HEK293 cells stably expressing the human P2X7 receptor (HEK293-hP2X7). Differences between the two antibiotics were assessed by monitoring the following: nucleotide-induced cytoplasmic free Ca2+ concentration changes, plasma membrane permeability changes, lactate dehydrogenase activity, cell morphology changes. Western blot and microscopic analyses of P2X7GFP-expressing cells were also performed. KEY RESULTS: In contrast to polymyxin B, the polymyxin B nonapeptide was unable to potentiate: a) the ATP-induced Ca2+ increase, b) pore formation and consequently ATP-mediated plasma membrane permeabilization; c) ATP-dependent cytotoxicity. Moreover, in contrast to polymyxin B, polymyxin B nonapeptide did not affect aggregation of the P2X7 receptor subunits and it did not potentiate P2X7-dependent cell fusion. CONCLUSIONS AND IMPLICATIONS: The effects of polymyxin B depended on the presence of its N-terminal fatty amino acid 6-methylheptanoic/octanoic-Dab residue as deletion of this residue abolished polymyxin B-dependent modulation of ATP-triggered responses. These findings are important in the search for allosteric modulators of the P2X7 receptor.

Adenosine Triphosphate↗
PubMed2006

Seeking a mechanism of action for the novel anticonvulsant lacosamide.

Lacosamide (LCM) is anticonvulsant in animal models and is in phase 3 assessment for epilepsy and neuropathic pain. Here we seek to identify cellular actions for the new drug and effects on recognised target sites for anticonvulsant drugs. Radioligand binding and electrophysiology were used to study the effects of LCM at well-established mammalian targets for clinical anticonvulsants. 10 microM LCM did not bind with high affinity to a plethora of rodent, guinea pig or human receptor sites including: AMPA; Kainate; NMDA (glycine/PCP/MK801); GABA(A) (muscimol/benzodiazepine); GABA(B); adenosine A1,2,3; alpha1, alpha2; beta1, beta2; M1,2,3,4,5; H1,2,3; CB1,2; D1,2,3,4,5; 5HT1A,1B,2A,2C,3,5A,6,7 and KATP. Weak displacement (25%) was evident at batrachotoxin site 2 on voltage gated Na+ channels. LCM did not inhibit neurotransmitter transport mechanisms for norepinephrine, dopamine, 5-HT or GABA, nor did it inhibit GABA transaminase. LCM at 100 microM produced a significant reduction in the incidence of excitatory postsynaptic currents (EPSC's) and inhibitory postsynaptic currents (IPSC's) in cultured cortical cells and blocked spontaneous action potentials (EC50 61 microM). LCM did not alter resting membrane potential or passive membrane properties following application of voltage ramps between -70 to +20 mV. The voltage-gated sodium channel (VGSC) blocker phenytoin potently blocked sustained repetitive firing (SRF) but, in contrast, 100 microM LCM failed to block SRF. No effect was observed on voltage-clamped Ca2+ channels (T-, L-, N- or P-type). Delayed-rectifier or A-type potassium currents were not modulated by LCM (100 microM). LCM did not mimic the effects of diazepam as an allosteric modulator of GABA(A) receptor currents, nor did it significantly modulate evoked excitatory neurotransmission mediated by NMDA or AMPA receptors (n > or = 5). Evidently LCM perturbs excitability in primary cortical cultures but does not appear to do so via a high-affinity interaction with an acknowledged recognition site on a target for existing antiepileptic drugs.

Acetamides↗
PubMed2006

On the mechanism of alleviation by phenobarbital of the malfunction of an epilepsy-linked GABA(A) receptor.

A mechanism for the alleviation of the malfunction of a mutated (gamma2(K289M)) epilepsy-linked gamma-aminobutyric acid (GABA) neurotransmitter receptor by phenobarbital is presented. Compared to the wild-type receptor, the GABA-induced current is considerably reduced in the mutated (alpha1beta2gamma2(K289M)) epilepsy-linked GABA(A) receptor [Baulac, S., Huberfeld, G., Gurfinkel-An, I., Mitropoulou, G., Beranger, A., Prud'homme, J. F., Baulac, M., Brice, A., Bruzzone, R., and LeGuer, E. (2001) Nat. Genet. 28, 46-48]. This is due to an impaired GABA-induced equilibrium between the closed- and open-channel forms of the receptor [Ramakrishnan, L., and Hess, G. P. (2004) Biochemistry 43, 7534-7540]. We report that a barbiturate anticonvulsant, phenobarbital, alleviates the effect of this mutation. Transient kinetic techniques with a millisecond-to-microsecond time resolution and the wild-type and mutated receptors recombinantly expressed in mammalian HEK293T cells were used. The efficacy of phenobarbital in potentiating currents elicited by a saturating concentration of GABA is about 3 times higher for the mutated receptor than for the wild type. The results indicate that phenobarbital alleviates the malfunction of the mutated receptor by increasing its channel-opening equilibrium constant (phi(-1) = k(op)/k(cl)) by about an order of magnitude. Phenobarbital changes the channel-opening rate constant (k(op)) by less than 2-fold but decreases the channel-closing rate constant (k(cl)) 8-fold. The dissociation constant of GABA is unaffected. The experiments also indicate that at saturating concentrations of GABA the mutated (gamma2(K289M)) form of the alpha1beta2gamma2 GABA(A) receptor is well suited for a rapid and simple screening of positive allosteric modulators of the receptor.

Allosteric Regulation↗
PubMed1978

The susceptibility of muscle phosphorylases a and b to digestion by a neutral proteinase from rat intestinal muscle. Comparison with the effects produced by pancreatic trypsin and chymotrypsin.

1. Phosphorylase b was inactivated three times more rapidly than phosphorylase a by a neutral, trypsin-like proteinase from rat intestinal muscle. Digestion of phosphorylase a produced a modified form which was deactivated by AMP. Removal of the pyridoxal phosphate cofactor increased the rate of inactivation of the b form by about 3-fold but the subceptibility of apophosphorylase a was no different from the holo form. 2. The extent of proteolysis of both holoenzyme forms, as guaged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, was limited and similar digestion patterns were obtained in both cases. 3. With (32)P-labelled phosphorylase a as substrate, the initial event in the inactivation was the release of a trichloroacetic acid-soluble peptide from the N-terminus of the enzyme, leaving the original 100000 subunit form essentially unchanged. Subsequent proteolysis was restricted, producing derivatives of mol.wt. 85000, 70000 and 65000, none of which contained any radioactive label. 4. By treatment of inactivated phosphorylase b with carboxypeptidase B, it was shown that the intestinal muscle proteinase had cleaved approximately 3 -Lys-X and 3 -Arg-X bonds in the polypeptide. 5. The protective effects of various allosteric modulators of phosphorylase on the inactivation of the a and b forms were generally in agreement with the known roles of the modifiers. Glucose increased the susceptibility of phosphorylase a. 6. Inactivation of phosphorylase b by trypsin and chymotrypsin also resulted in limited proteolysis but, in both cases, the digestion patterns obtained on sodium dodecyl sulphate/polyacrylamide gels were different from each other and from the pattern obtained with the intestinal muscle proteinase. 7. Inactivation of phosphorylase b by the muscle proteinase is about 100 times more rapid than the effects produced by trypsin or chymotrypsin when the activities are compared on an equimolar basis. 8. Consideration is given to regulation of the rate of enzyme degradation intracellularly by modulation of the conformation and susceptibility of the enzyme via factors such as covalent modification, allosteric ligands and state of aggregation.

Adenosine Monophosphate↗
PubMed2004

Functional and structural analysis of the GABAA receptor alpha 1 subunit during channel gating and alcohol modulation.

The substituted cysteine accessibility method has proven useful for investigating structural changes of the gamma-aminobutyric acid type A (GABA(A)) receptor during channel gating and allosteric modulation. In the present study, the surface accessibility and reaction rate of propyl- and hexyl-methanethiosulfonate to cysteine residues introduced into the third transmembrane segment of the GABA(A) receptor alpha(1) subunit were examined. GABA-induced currents in Xenopus oocytes expressing wild type and cysteine mutant GABA(A) receptors were recorded before and after application of methanethiosulfonate (MTS) reagents in the resting, GABA- or alcohol-bound (ethanol or hexanol) states. Our results indicate that a water-filled cavity exists around the Ala(291) and Tyr(294) residues of the third transmembrane segment, in agreement with previous results. Furthermore, our data indicate that a conformational change produced by alcohols (200 mM ethanol or 0.5 mM hexanol) exposure induces the water cavity around the A291C and Y294C residues to extend deeper, causing the A295C and F296C residues to become accessible to the MTS reagents. In addition, exposure of the A291C, Y294C, F296C, and V297C mutants to MTS reagents in the presence of GABA had significant effects on their GABA-induced currents, indicating that the water cavity around A291C and Y294C residues expanded to F296C and V297C by a structural movement caused by GABA binding. Our data show that GABA(A) receptor is a dynamic protein during alcohol modulation and channel gating.

Allosteric Regulation↗
PubMed1996

Delta subunit inhibits neurosteroid modulation of GABAA receptors.

Neurosteroid modulation of GABAA receptors has been observed with all subunit combinations investigated; however, hetero-oligomeric GABAA receptors containing delta subunits were not studied previously. We describe the effect of delta subunit expression on 3alpha,21-dihydroxy-5alpha-pregnan-20-1 (THDOC)-induced potentiation of GABA-gated currents in transfected HEK 293 cells and in cerebellar granule cells in vitro. THDOC (100 nM) significantly potentiated GABA-gated currents in cells transfected with combinations of alpha1, alpha6, beta3, and gamma2 subunit cDNAs, whereas cotransfection of delta subunit cDNA inhibited this potentiation. In contrast, the direct Cl- channel activation by THDOC at higher concentrations (1-10 microM) was not significantly dependent on delta subunit cotransfection. These results suggest that the presence of the delta subunit inhibits GABAA receptor modulation but not the direct activation by neurosteroids. Cotransfection with delta subunit also affected the negative allosteric modulation by pregnenolone sulfate. THDOC potentiation of GABA-gated currents was greater in cerebellar granule cell cultures at 4 d in vitro (DIV) compared with those at 14 DIV. Single-cell reverse transcription-PCR analysis of the mRNAs expressed in cultured cerebellar granule cells shows that an increased number of granule cells at 14 DIV express delta subunit mRNAs as compared with 4 DIV granule cells. The presence of delta subunit mRNAs detected in individual cells correlated well with the lack of sensitivity to THDOC. These results suggest that developmental expression of GABAA receptor delta subunits may play an important role in determining the region-specific neurosteroid-induced modification of fast inhibitory synaptic function.

Allosteric Regulation↗
PubMed2000

Forskolin modulates acetylcholine receptor gating by interacting with the small extracellular loop between the M2 and M3 transmembrane domains.

1. Forskolin acts as an allosteric modulator of muscle-type nicotinic acetylcholine receptors. Receptors from mouse muscle and Torpedo electroplax demonstrate differential sensitivity to inhibition by forskolin. Previous work from this laboratory suggested that the gamma subunit is responsible for this differential sensitivity. 2. We have used a series of mouse/Torpedo species-chimeric gamma subunits to further define the site of forskolin interaction with the gamma subunit. Analysis of the patterns of forskolin inhibition of receptors containing mouse/Torpedo chimeric gamma subunits along with the mouse alpha, beta, and delta subunits suggests that forskolin interacts with the small extracellular domain that links the M2 and M3 transmembrane domains (the M2-M3 linker). 3. We suggest that the M2-M3 linker domain plays an important role in the transduction of ligand binding to the conformational changes that result in channel opening.

Allosteric Regulation↗
PubMed1995

Neuroactive steroid modulation of [3H]muscimol binding to the GABAA receptor complex in rat cortex.

Epalons are a subclass of neuroactive steroids which are positive allosteric modulators of the GABAA receptor acting via a unique site on the receptor complex. Enhancement of [3H]muscimol binding to the GABA recognition site was observed to be either full (> 150%) or limited (110-135%) and specific for epalons. Both one and two component modulation was observed. Saturation studies performed in the presence of 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha,5 alpha-P) showed that 3 alpha, 5 alpha-P increased the density of high affinity [3H]muscimol sites and doubled the affinity of low affinity sites. 3 alpha,5 alpha-P had no effect on the affinity of the high affinity site or the density of the low affinity site. These data indicate that: (1) epalons are potent, stereoselective enhancers of [3H]muscimol binding; (2) epalons display varying levels of efficacy and some exhibit two-component enhancement; and (3) 3 alpha,5 alpha-P enhancement of [3H]muscimol binding results from both an increased density of high affinity sites and an increased affinity for low affinity sites.

Anesthetics↗
PubMed1999

gamma-Aminobutyrate-A receptor modulation by 3-aryl-1-(arylsulfonyl)- 1,4,5,6-tetrahydropyridazines.

A series of 3-aryl-1-(arylsulfonyl)-1,4,5,6-tetrahydropyridazine allosteric modulators of the GABAA receptor was synthesized, and biological activity was examined in vitro and in vivo. Beginning with 1a, stepwise modification of the substituents and conservation of the scaffold yielded a chemical series in which the modulatory activity was enhanced by the presence of GABA. The SAR suggests, but does not establish, that the compounds bind to the steroid binding site on the GABAA receptor. The GABA shift for each compound indicates that all compounds in this series are either agonists or partial agonists.

Animals↗
PubMed1994

3 alpha-Hydroxy-5 beta-pregnan-20-one sulfate: a negative modulator of the NMDA-induced current in cultured neurons.

We have shown previously that the neurosteroid pregnenolone sulfate acts as a positive allosteric modulator at the N-methyl-D-aspartate (NMDA) receptor while inhibiting the kainate, the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), the glycine, and the gamma-aminobutyric acid (GABA) responses of chick spinal cord neurons. Here, we report that 3 alpha-hydroxy-5 beta-pregnan-20-one sulfate (5 beta 3 alpha S), a sulfated form of naturally occurring 5 beta 3 alpha, inhibits both the NMDA and the non-NMDA receptor-mediated responses as measured by whole cell voltage clamp recordings. 100 microM 5 beta 3 alpha S rapidly and reversibly inhibits the response to 30 microM NMDA by 66%, 50 microM kainate by 37%, and 25 microM AMPA by 29%. Application of 50 microM nonsulfated 5 beta 3 alpha does not produce any significant effect on the NMDA response, demonstrating that the sulfate moiety is important for the effect of 5 beta 3 alpha S on the NMDA response. The effect of 5 beta 3 alpha S on the NMDA response is concentration dependent, with an EC50 of 62 microM. 5 beta 3 alpha S reduces the maximum NMDA response with little effect on the NMDA EC50, indicating that antagonism of the NMDA response by 5 beta 3 alpha S is noncompetitive. The fact that 5 beta 3 alpha S inhibition of the NMDA response is neither agonist nor voltage dependent demonstrates that 5 beta 3 alpha S does not act as an open channel blocker. Furthermore, inhibition of the NMDA response by 5 beta 3 alpha S is not reduced by the addition of a maximal concentration (10 microM) of glycine, indicating that 5 beta 3 alpha S does not act via the glycine recognition site. The inhibitory action of 5 beta 3 alpha S on the NMDA and non-NMDA receptors may provide a basis for inhibiting glutamate receptor-induced seizures and excitotoxic cell death.

Animals↗
PubMed2006

Identification of a Zn2+-binding site on the dopamine D2 receptor.

Zinc (II) modulates the function of many integral membrane proteins. To identify the Zn(2+)-binding site responsible for allosteric modulation of the D(2) dopamine receptor, we first demonstrated that the binding site is likely located in extracellular loops or in transmembrane regions that are accessible from the extracellular milieu. We mutated every histidine in these regions to alanine; two mutants, H394A and H399A, exhibited a reduced response to Zn(2+). Combined mutation of H394 and H399 caused a larger effect of zinc than did either single mutation. Mutation of other potential Zn(2+)-binding residues predicted to be in proximity to H394 or H399 did not substantially alter the potency of Zn(2+). The double mutant H394A/H399A was similar to D(2) in affinity for [(3)H]spiperone and ability to inhibit cyclic AMP accumulation. We conclude that binding of Zn(2+) to H394 and H399 on the dopamine D(2) receptor contributes to allosteric regulation of antagonist binding.

Amino Acid Substitution↗
PubMed1989

Differential effects of iodide and chloride on allosteric interactions of the GABAA receptor.

t-[35S]Butylbicyclophosphorothionate [( 35S]TBPS) has been shown to bind to the GABAA receptor complex. The binding is modulated allosterically by drugs that interact at components of the receptor complex. The present studies were designed to evaluate the influence of ionic environment and state of equilibrium on the allosteric modification of [35S]TBPS binding. In both I- and Cl- under nonequilibrium conditions, diazepam, gamma-aminobutyric acid (GABA), and pentobarbital (PB) stimulate and methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) inhibits [35S]TBPS binding. In addition, there is an inhibitory component to the effect of GABA and PB at higher drug concentrations. These effects are blocked by the appropriate antagonists for each drug. In Cl-, the stimulation of [35S]TBPS binding by drugs disappears at equilibrium, whereas the inhibition by GABA and PB persists. The inhibitory effect of DMCM in Cl- also disappears at equilibrium. When assayed in I- at equilibrium, however, DMCM stimulates [35S]TBPS binding. In addition, bicuculline, which is without effect under nonequilibrium conditions in either Cl- or I-, stimulates [35S]TBPS binding in I- at equilibrium. The persistent effects of DMCM, bicuculline, and GABA in I- are accompanied by alterations in the affinity of [35S]TBPS for its receptor. In addition, the stimulation of [35S]TBPS binding by GABA is associated with a decreased number of [35S]TBPS binding sites. These data demonstrate that receptor complex interactions with anions influence the responsiveness to drug binding.

Allosteric Regulation↗
PubMed1989

Distribution of a putative endogenous modulator of the GABAergic system in human brain.

Diazepam binding inhibitor (DBI) is a novel neuropeptide purified from rat, cow, and human brain that allosterically modulates GABAergic transmission by binding to benzodiazepine (BDZ)-recognition sites. Using a specific radioimmunoassay for human DBI, we investigated the distribution of this peptide in different brain areas. We characterized with high-pressure liquid chromatography the DBI immunoreactivity in brain tissue obtained by biopsy and autopsy; we detected one molecular species of DBI in both instances. The regional distribution of DBI in the human brain is similar to that observed in rat brain: high concentrations in cortical and limbic areas, cerebellum, and brainstem, and low concentrations in the basal ganglia. These data suggest a modulatory role for DBI in human brain.

Brain↗
PubMed1983

Some comparative properties of pyruvate kinase in haematopoietic cells and erythrocytes from rainbow trout (Salmo gairdneri R).

1. Temperature acts as a pyruvate kinase regulator in haematopoietic cells and erythrocytes. 2. Fructose-1,6-biphosphate and alanine act as allosteric modulators of pyruvate kinase in haematopoietic cells, while in erythrocytes although fructose-1,6-biphosphate exerts also allosteric effect, alanine appears to be a competitive inhibitor. ATP (1.0 mM) does not exert any clear effect on pyruvate kinase of both cellular populations. 3. The level of specific activity of pyruvate kinase in haematopoietic cells is 40-fold that of PK from erythrocytes.

Adenosine Triphosphate↗
PubMed1998

Two imidazoquinoxaline ligands for the benzodiazepine site sharing a second low affinity site on rat GABA(A) receptors but with the opposite functionality.

1. Imidazoquinoxaline PNU-97775 and imidazoquinoline PNU-101017 are benzodiazepine site ligands with a second low affinity binding site on GABA(A) receptors, the occupancy of which at high drug concentrations reverses their positive allosteric activity via the benzodiazepine site, and may potentially minimize abuse liability and physical dependence. 2. In this study we discovered, with two imidazoquinoxaline analogues, that the functionality of the second site was altered by the nitrogen substituent on the piperazine ring moiety: PNU-100076 with a hydrogen substituent on the position produced a negative allosteric effect via the second low affinity site, like the parent compounds, while PNU-100079 with a trifluoroethyl substituent produced a positive allosteric response. 3. These functional characteristics were monitored with Cl- currents measurements in cloned rat alphaxbeta2gamma2 subtypes of GABA(A) receptors expressed in human embryonic kidney 293 cells, and further confirmed in rat cerebrocortical membranes containing complex subtypes of GABA(A) receptors with binding of [35S]-TBPS, which is a high affinity ligand specific for GABA(A) receptors with exquisite sensitivity to allosteric modulations. 4. This structure-functional relationship could be exploited to further our understanding of the second allosteric site of imidazoquinoxaline analogues, and to develop more effective benzodiazepine site ligands without typical side effects associated with those currently available on the market.

Animals↗
PubMed2000

Functional impact of the ryanodine receptor on the skeletal muscle L-type Ca(2+) channel.

L-type Ca(2+) channel (L-channel) activity of the skeletal muscle dihydropyridine receptor is markedly enhanced by the skeletal muscle isoform of the ryanodine receptor (RyR1) (Nakai, J., R.T. Dirksen, H. T. Nguyen, I.N. Pessah, K.G. Beam, and P.D. Allen. 1996. Nature. 380:72-75.). However, the dependence of the biophysical and pharmacological properties of skeletal L-current on RyR1 has yet to be fully elucidated. Thus, we have evaluated the influence of RyR1 on the properties of macroscopic L-currents and intracellular charge movements in cultured skeletal myotubes derived from normal and "RyR1-knockout" (dyspedic) mice. Compared with normal myotubes, dyspedic myotubes exhibited a 40% reduction in the amount of maximal immobilization-resistant charge movement (Q(max), 7.5 +/- 0.8 and 4.5 +/- 0.4 nC/muF for normal and dyspedic myotubes, respectively) and an approximately fivefold reduction in the ratio of maximal L-channel conductance to charge movement (G(max)/Q(max)). Thus, RyR1 enhances both the expression level and Ca(2+) conducting activity of the skeletal L-channel. For both normal and dyspedic myotubes, the sum of two exponentials was required to fit L-current activation and resulted in extraction of the amplitudes (A(fast) and A(slow)) and time constants (tau(slow) and tau(fast)) for each component of the macroscopic current. In spite of a >10-fold in difference current density, L-currents in normal and dyspedic myotubes exhibited similar relative contributions of fast and slow components (at +40 mV; A(fast)/[A(fast) + A(slow)] approximately 0.25). However, both tau(fast) and tau(slow) were significantly (P < 0.02) faster for myotubes lacking the RyR1 protein (tau(fast), 8.5 +/- 1.2 and 4.4 +/- 0.5 ms; tau(slow), 79.5 +/- 10.5 and 34.6 +/- 3.7 ms at +40 mV for normal and dyspedic myotubes, respectively). In both normal and dyspedic myotubes, (-) Bay K 8644 (5 microM) caused a hyperpolarizing shift (approximately 10 mV) in the voltage dependence of channel activation and an 80% increase in peak L-current. However, the increase in peak L-current correlated with moderate increases in both A(slow) and A(fast) in normal myotubes, but a large increase in only A(fast) in dyspedic myotubes. Equimolar substitution of Ba(2+) for extracellular Ca(2+) increased both A(fast) and A(slow) in normal myotubes. The identical substitution in dyspedic myotubes failed to significantly alter the magnitude of either A(fast) or A(slow). These results demonstrate that RyR1 influences essential properties of skeletal L-channels (expression level, activation kinetics, modulation by dihydropyridine agonist, and divalent conductance) and supports the notion that RyR1 acts as an important allosteric modulator of the skeletal L-channel, analogous to that of a Ca(2+) channel accessory subunit.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed2005

The existence of a second allosteric site on the M1 muscarinic acetylcholine receptor and its implications for drug design.

Fully flexible docking of KT5720, an allosteric modulator of the muscarinic receptors, was performed on a dynamic model of the M(1) muscarinic acetylcholine receptor. The results confirmed the existence of a second allosteric site, located on the intracellular face of the receptor. These results would be beneficial for the design of modulators of this receptor to be used as an effective alternative against the Alzheimer's disease.

Allosteric Site↗