A model for the calmodulin-peptide complex based on the troponin C crystal packing and its similarity to the NMR structure of the calmodulin-myosin light chain kinase peptide complex.

In the crystal structure of troponin C, the holo C-domain is bound in a head-to-tail fashion to the A-helix of the apo N-domain of a symmetry-related molecule. Using this interaction, we have proposed a model for the calmodulin-peptide complex. We find that the interaction of the C-domain with the A-helix is similar to that observed in the NMR structure of the calmodulin-myosin light chain kinase (MLCK) peptide complex. This similarity in binding has enabled us to make a precise sequence alignment of the target peptides in the calmodulin-binding cleft and to rationalize the amino acid sequence-dependent binding strengths of various peptides. Our model differs from that proposed by Strynadka and James (Proteins Struct. Funct. Genet. 7, 234-248, 1990) in that the peptides are rotated by 100 degrees in the calmodulin binding cleft.     

Calcium/Calmodulin Inhibits the Binding of Specific [125I]Omega-Conotoxin GVIA to Chick Brain Membranes

The effect of Ca²⁺/calmodulin (CaM) on the specific binding of [¹²⁵I]omega-conotoxin GVIA (¹²⁵I--CTX) to crude membranes from chick brain was investigated. When we examined the effects of the activation of various endogenous protein kinases on specific [¹²⁵I]-CTX binding to crude membranes, we observed that Ca²⁺/CaM had an inhibitory effect regardless of whether or not the standard medium contained ATP (0.5 mM). Ca²⁺/CaM also had an inhibitory effect in a simple binding-assay medium containing HEPES-HCl buffer, BSA, Ca²⁺ and CaM, and this effect was dependent on the concentration of Ca²⁺. The effect of Ca²⁺/CaM was attenuated by the CaM antagonists W-7 and CaM-kinase II fragment (290–309). An experiment with modified ELISA using purified anti -CTX antibody indicated that Ca²⁺/CaM did not affect the direct binding of [¹²⁵I]-CTX and CaM. These results suggest that Ca²⁺/CaM either directly or indirectly affects specific [¹²⁵I]-CTX binding sites, probably N-type Ca²⁺ channels in crude membranes from chick whole brain.     

A rendezvous with the queen of ion channels: Three decades of ion channel research by David T Yue and his Calcium Signals Laboratory

David T. Yue was a renowned biophysicist who dedicated his life to the study of Ca²⁺ signaling in cells. In the wake of his passing, we are left not only with a feeling of great loss, but with a tremendous and impactful body of work contributed by a remarkable man. David's research spanned the spectrum from atomic structure to organ systems, with a quantitative rigor aimed at understanding the fundamental mechanisms underlying biological function. Along the way he developed new tools and approaches, enabling not only his own research but that of his contemporaries and those who will come after him. While we cannot hope to replicate the eloquence and style we are accustomed to in David's writing, we nonetheless undertake a review of David's chosen field of study with a focus on many of his contributions to the calcium channel field.     

Binding of3H-phenamil,an irreversible amiloride analog,to toad urinary bladder: effects of aldosterone and vasopressin

Summary Phenamil, an analog of amiloride, has previously been shown to bind specifically to sodium channels in toad bladder (J.L. Garvin et al.,J. Membrane Biol. 87:45–54, 1985). In this paper,³H-phenamil was used to measure sodium channel density in both isolated epithelial cells and intact bladders. From the specific binding to intact bladders, a channel density of 455±102 channels/m² was calculated. No correlation between specific binding and the magnitude of irreversible inhibition of shortcircuit current was found. Pretreatment of intact bladders with 1 mg/ml trypsin reduced specific binding to isolated cells by 82±5%. In isolated cells, neither aldosterone nor vasopressin had any significant effect on specific phenamil binding. It is inferred that phenamil binds to both open and closed channels which may be either in the mucosal membrane or in the submembrane space. Finally, and rather surprisingly, we found that³H-phenamil binds irreversibly to the basolateral membrane at concentrations as low as 4×10^–7 m. Therefore, care must be used in interpreting binding studies with amiloride or its analog at such concentrations.     

Ligands Binding and Molecular Simulation: the Potential Investigation of a Biosensor Based on an Insect Odorant Binding Protein

Based on mimicking biological olfaction, biosensors have been applied for the detection of various ligands in complex environment, which could represent one of the most promising research fields. In this study, the basic characters of one insect odorant binding protein (OBP) as a biosensor were explored. To explore the molecular recognition process, the tertiary structure of the protein was modeled and the protein-ligand interactions with 1,536,550 chemicals were investigated by the molecular docking. The availability of large amount of recombinant SlitOBP1 overcame the difficulty to obtain biological sensing material. After obtained the purified recombinant protein, the result of fluorescence binding assays proved the candidate protein has good affinities with the majority of the tested chemicals. With the aid of simulation docking, the key conserved amino acids within the binding site were identified and then mutated to alanine. After mutation, the protein-ligand binding characteristics were recorded, and the competitive binding assays were carried out to provide experimental verification. The detailed information on its structure and affinities investigated in this study could allow the design of specific mutants with desired characteristics, which provides a solid base for tailoring OBP for biosensor and provides a role model for screening the other elements in olfactory system for different applications.     

On the other hand: The stereoselectivity of drug action at ion channels

Ion channels are pharmacological receptors with specific drug binding sites. These binding sites define specific structure–function relationships for the actions of drug classes. Interpretation of these structure–function relationships may be complex because of state-dependent drug-channel interactions. These state-dependent interactions determine affinity and access of drug to binding sites and may result in both quantitative and qualitative changes in structure–function relationships including stereoselectivity. A channel-active drug may exhibit antagonist or activator properties according to membrane potential and the stereoselectivity of interaction may also change with channel state. © 1994 Wiley-Liss, Inc.     

Chiral aspects of drug action at ion channels: a commentary on the stereoselectivity of drug actions at voltage-gated ion channels with particular reference to verapamil actions at the Ca2+ channel.

Ion channels may be considered as pharmacological receptors possessing specific drug binding sites with defined structure-activity relationships. Accordingly drug binding to ion channels is stereoselective. Interpretation of this stereoselectivity may be complex because of the existence of differences in affinity and access to different channel states. Such state-dependent interactions may give rise to quantitative and qualitative differences in stereoselectivity. The implications of such differences are reviewed for drug action at Na+, K+ and Ca2+ channels. Detailed attention is paid to the actions of verapamil enantiomers in the cardiovascular system where activities differ in vascular and cardiac tissues because of state-dependent interactions and stereoselective first-oass metabolism.     

Molecular basis of the interaction between IGFBP-3 and retinoid X receptor: role in modulation of RAR-signaling

IGFBP-3 interacts with the retinoid X receptor-alpha (RXRalpha) and retinoic acid receptor-alpha (RARalpha) and thereby interferes with the formation of RXR:RAR heterodimers. Here we identify the domains in RXRalpha and IGFBP-3 that participate in this interaction. When different regions of RXRalpha were expressed independently, we found that only the DNA-binding domain (C-domain) bound IGFBP-3. Residues in the second Zn-finger loop (Gln49, Arg52), which contribute to C-domain dimerization on DR1 response elements, proved essential to IGFBP-3 binding. In complementary studies, we found that residues within the N-terminal domain of IGFBP-3 (Thr58, Arg60) and motifs in its C-terminal domain ((220)LysLysLys, (228)LysGlyArgLysArg) were required for interaction with RXRalpha and RARalpha. Unlike wild-type IGFBP-3, the non-retinoid receptor-binding mutants of IGFBP-3 were unable to attenuate all-trans-retinoic acid-induced transactivation of the RAR response element by RXR:RAR heterodimers. We conclude that residues in both the N- and C-terminal domains of IGFBP-3 are involved in binding the retinoid receptors, and that this interaction is essential to the modulation of RAR-signaling by IGFBP-3.     

The differential effects of single or repeated restraint stress on kainic acid-induced neuronal death in the hippocampal CA3 region: the role of glucocorticoid and various signal molecules

The effect of stress mediators following the stress period and addition time is a controversial issue until now. Thus, we aim to clarify the differential effects of single restraint stress (SS) or repeated restraint stress (RS) on kainic acid (KA)-induced neuronal death especially as addressing not only the role of glucocorticoid (Gc) and its receptor but also the signal pathway leading to cAMP response element binding protein phosphorylation (pCREB) and its functional role during stress. In the present study, we found that although RS did not show any difference on serum Gc level and hippocampal Gc receptor level compared to SS, SS exacerbated KA-induced neuronal death in hippocampal CA3 region, but RS did not. Moreover, pre-treatment with RU 38486 (Gc receptor antagonist) abolished the effect of SS on KA-induced neuronal death without an effect on KA toxicity itself. Furthermore, RS aggravates KA-induced neuronal death when CREB phosphorylation was deprived by KN-93 (calcium/calmodulin-dependent protein kinase II inhibitor). However, other signal molecules inhibitors such as PD98059 (MEK1/2 inhibitor) and SP600125 (p-p38 inhibitor) have no effect on KA-induced neuronal death after RS although these signal molecule were increased during SS or RS. These findings suggest that pCREB expression via calcium/calmodulin-dependent protein kinase II phosphorylation during RS comprise one of the balancers against Gc induced by stress.     

Binding of the respiratory chain inhibitor antimycin to the mitochondrial bc1 complex: a new crystal structure reveals an altered intramolecular hydrogen-bonding pattern

Antimycin A (antimycin), one of the first known and most potent inhibitors of the mitochondrial respiratory chain, binds to the quinone reduction site of the cytochrome bc1 complex. Structure-activity relationship studies have shown that the N-formylamino-salicyl-amide group is responsible for most of the binding specificity, and suggested that a low pKa for the phenolic OH group and an intramolecular H-bond between that OH and the carbonyl O of the salicylamide linkage are important. Two previous X-ray structures of antimycin bound to vertebrate bc1 complex gave conflicting results. A new structure reported here of the bovine mitochondrial bc1 complex at 2.28 A resolution with antimycin bound, allows us for the first time to reliably describe the binding of antimycin and shows that the intramolecular hydrogen bond described in solution and in the small-molecule structure is replaced by one involving the NH rather than carbonyl O of the amide linkage, with rotation of the amide group relative to the aromatic ring. The phenolic OH and formylamino N form H-bonds with conserved Asp228 of cytochrome b, and the formylamino O H-bonds via a water molecule to Lys227. A strong density, the right size and shape for a diatomic molecule is found between the other side of the dilactone ring and the alphaA helix.     

Biochemical and Pharmacological Characterization of [3H]GBR 12935 Binding In Vitro to Rat Striatal Membranes: Labeling of the Dopamine Uptake Complex

Abstract: Binding of the selective dopamine (DA) uptake inhibitor [³H]GBR 12935 to rat striatal membranes was characterized biochemically and pharmacologically. [³H]-GBR 12935 binding at 0°C was reversible and saturable and Scatchard analysis indicated a single binding site with a K_D of 5.5 nM and a B_max of 760 pmol/mg tissue. [³H]GBR 12935 labeled two binding sites. One binding site was identified as the classic DA uptake site, since methylphenidate, cocaine, diclofensine, and Lu 19–005 potently inhibited [³H]GBR 12935 binding to it. Binding to the second site was inhibited by high concentrations of the above compounds. IC₅₀ values for inhibition of [³H]GBR 12935 binding to the DA uptake site were proportional to IC50 values for inhibition of DA uptake. However, substrates of DA uptake, e.g., DA and 1-methyl-4-phenylpyridine, and DA releasers, e.g., the amphetamines, inhibited [³H]GBR 12935 binding less than DA uptake. Rate experiments excluded the possibility that these “weak” inhibitors affected the binding by alloste-ric coupled binding sites. The second binding site was not a noradrenergic, serotonergic, or GABAergic uptake site. Neither was it a dopaminergic, acetylcholinergic, histaminic, serotonergic, or adrenergic receptor. However, [³H]GBR 12935 was potently displaced from it by disubstituted piper-azine derivatives, i.e., flupentixol and piflutixol. DA uptake and the DA uptake binding site of [³H]GBR 12935 were located primarily in the striatum, but the piperazine acceptor site was distributed uniformly throughout the brain. Also only the DA uptake binding site was destroyed by 6-OH-DA. Thus, [³H]GBR 12935 labels the classic DA uptake site in rat striatum and also a piperazine acceptor site. Substrates for DA uptake and releasers of DA inhibited [³H]GBR 12935 binding with low potency, but did not alter the rate constants for [³H]GBR 12935 binding. Therefore inhibitors of DA uptake label the carrier site and prevent the carrier process.     

Labelling of "Peripheral-Type" Benzodiazepine Binding Sites in the Rat Brain By Using [3H]PK 11195, an Isoquinoline Carboxamide Derivative: Kinetic Studies and Autoradiographic Localization

PK 11195 [1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide] is a new ligand for the "peripheral-type" benzodiazepine binding sites, chemically unrelated to benzodiazepines. It displaces with a very high potency (IC50 congruent to 10(-9) M) [3H]-RO5-4864 (a benzodiazepine which specifically labels the peripheral-type sites) from its binding sites. [3H]PK 11195 binds to a membrane fraction from rat brain cortex and rat olfactory bulb in a saturable and reversible manner with a very high affinity (KD = 10(-9) M). The number of maximal binding sites was ten times greater in the olfactory bulb than in the brain cortex. The order of potency of several compounds as displacers at 25 degrees C (PK 11195 greater than RO5-4864 greater than diazepam greater than dipyridamole greater than clonazepam) demonstrates that [3H]PK 11195 binds to the peripheral-type benzodiazepine binding sites. The KD value for the [3H]PK 11195 binding is not affected by temperature changes, whereas RO5-4864 and diazepam affinities decrease with increasing temperatures. Autoradiographic images of [3H]PK 11195 binding to rat brain sections show that binding sites are mainly localized in the olfactory bulb, median eminence, choroid plexus, and ependyma. This ligand could be a useful tool to elucidate the physiological and pharmacological relevance of these binding sites.     

Modification of [3H]MK801 Binding to Rat Brain NMDA Receptors after the Administration of a Convulsant Drug and an Adenosine Analogue: A Quantitative Autoradiographic Study

Specific [³H]MK801 binding to rat brain NMDA receptors after the administration of the convulsant drug 3-mercaptopropionic acid (MP) and the adenosine analogue cyclopentyladenosine (CPA) was studied by means of a quantitative autoradiographic method. MP administration (150 mg/kg, i.p.) caused significant decreases in [³H]MK801 binding in several hippocampus subareas and layers, mainly in CA1 and CA3 at seizure (11–27%) and postseizure (8–16%) and in cerebral occipital cortex at seizure (18–22%). In nucleus accumbens, a rise was observed at postseizure (44%) and a tendency to increase at seizure (24%). CPA (2mg/kg, i.p.) decreased ligand binding in hippocampus (CA1, CA2, CA3) (17–22%) and in occipital cerebral cortex (18–24%). When CPA was administered 30 minutes before MP (which delayed seizure onset) and rats were sacrified at seizure, decreases in [³H]MK801 binding in several layers of CA1 and CA3 of hippocampus (11–27%) and in CA1, CA2, CA3 (24–35%) after CPA+MP postseizure, and an increase in CA2 after CPA and CPA+MP postseizure (20–34%), were observed. A drop was found in the occipital subarea (18–24%) after CPA and in the frontal and occipital subarea after CPA+MP postseizure (24–34%) while no changes were observed in any treatment involving the other cerebral cortex regions, thalamic nuclei, caudate putamen and olfactory tubercle. These results show that [³H]MK801 binding changes according to drug treatment and the area being studied, thus indicating a different role in seizure activity.     

Synthesis of cyclooctapeptides: constraints analogues of the peptidic neurotoxin, ω‐agatoxine IVB—an experimental point of view

ω‐AGA IVB is an important lead structure when considering the design of effectors of glutamate release inducting P/Q‐type calcium channels. The best route to achieve the analogues possessing the three‐dimensional arrangement corresponding to the native binding loop was the introduction of constraint by ring formation via side chain to side chain lactamization for suitably protected Lys and Glu residues. Since tryptophane residue located at position 14 of this neuropeptide has been suggested as essential for binding, analogues in which this amino acid was replaced by aza‐tryptophane and alanine were synthesized. The synthesis was carried out on various acid‐labile resins (BARLOS chlorotrityl, Rink amide, PEG‐based or Wang resins), by Fmoc strategy. In this paper, we describe optimization of the peptide cyclization with various protecting groups, and on resin or in solution cyclization experimental parameters. Copyright © 2007 European Peptide Society and John Wiley & Sons, Ltd.     

Roles for Nitric Oxide as an Intra- and Interneuronal Messenger at NMDA Release-Regulating Receptors: Evidence from Studies of the NMDA-Evoked Release of [3H]Noradrenaline and d-[3H]Aspartate from Rat Hippocampal Slices

Abstract: N-Methyl-d -aspartate (NMDA) receptors regulating the release of [³H]noradrenaline ([³H]NA) and d -[³H]aspartate (d -[³H]Asp) were investigated in superfused slices of rat hippocampus in the presence and absence of nitrergic drugs to examine a possible role for nitric oxide (NO) in the release process. In Mg²⁺-free Krebs-Henseleit buffer, the NMDA-evoked release of [³H]NA and d -[³H]Asp was Ca²⁺ dependent and inhibited by the NMDA antagonist (±)-3-(2-carboxypiperazin-4-yl)propenyl-1-phosphonic acid. NMDA-stimulated release of [³H]NA was tetrodotoxin (TTX; 0.1–2 µM) sensitive, whereas that for d -[³H]Asp was TTX insensitive, indicating that the NMDA receptors involved are differentially localized; those for d -[³H]Asp appear to be presynaptic, whereas those for [³H]NA are extrasynaptic in location. l -Arginine (100 µM), the natural precursor of NO synthesis, enhanced NMDA-evoked release of [³H]NA (100%) and d -[³H]Asp (700%). Exogenous NO donors—sodium nitroprusside, 3-morpholinosyndnomine, and S-nitroso-N-acetylpenicillamine (all 100 µM)—stimulated the NMDA-evoked release. An exception was the inhibition by nitroprusside of NMDA-evoked release of [³H]NA, where the presence of antioxidants may influence channel activity. Inhibitors of NO synthase (N^G-nitro-, N^G-methyl-, and N^G-amino-l -arginine, all 100 µM) attenuated (50–80%) the NMDA-stimulated release of [³H]NA and d -[³H]Asp, as did KN-62 (10 µM), a specific inhibitor of calmodulin kinase II. Our data support roles for the NO transducing system subsequent to the activation of NMDA release-regulating receptors as both an intraneuronal (presynaptically) and an extraneuronal messenger.     

Interplay of Troponin- and Myosin-Based Pathways of Calcium Activation in Skeletal and Cardiac Muscle: The Use of W7 as an Inhibitor of Thin Filament Activation

To investigate the interplay between the thin and thick filaments during calcium activation in striated muscle, we employed n-(6-aminohexyl) 5-chloro-1-napthalenesulfonamide (W7) as an inhibitor of troponin C and compared its effects with that of the myosin-specific inhibitor, 2,3-butanedione 2-monoxime (BDM). In both skeletal and cardiac fibers, W7 reversibly inhibited ATPase and tension over the full range of calcium activation between pCa 8.0 and 4.5, resulting in reduced calcium sensitivity and cooperativity of ATPase and tension activations. At maximal activation in skeletal fibers, the W7 concentrations for half-maximal inhibition (K_I) were 70–80 μM for ATPase and 20–30 μM for tension, nearly >200-fold lower than BDM (20 mM and 5–8 mM, respectively). When W7 (50 μM) and BDM (20 mM) were combined in skeletal fibers, the ATPase and tension-pCa curves exhibited lower apparent cooperativity and maxima and higher calcium sensitivity than expected from two independent activation pathways, suggesting that the interplay between the thin and thick filaments varies with the level of activation. Significantly, the inhibition of W7 increased the ATPase/tension ratio during activation in both muscle types. W7 holds much promise as a potent and reversible inhibitor of thin filament-mediated calcium activation of skeletal and cardiac muscle contraction.     

Battling for Ribosomes: Translational Control at the Forefront of the Antiviral Response

In the early stages of infection, gaining control of the cellular protein synthesis machinery including its ribosomes is the ultimate combat objective for a virus. To successfully replicate, viruses unequivocally need to usurp and redeploy this machinery for translation of their own mRNA. In response, the host triggers global shutdown of translation while paradoxically allowing swift synthesis of antiviral proteins as a strategy to limit collateral damage. This fundamental conflict at the level of translational control defines the outcome of infection. As part of this special issue on molecular mechanisms of early virus–host cell interactions, we review the current state of knowledge regarding translational control during viral infection with specific emphasis on protein kinase RNA-activated and mammalian target of rapamycin-mediated mechanisms. We also describe recent technological advances that will allow unprecedented insight into how viruses and host cells battle for ribosomes.