The Experts below are selected from a list of 297 Experts worldwide ranked by ideXlab platform
Kazuki Sato - One of the best experts on this subject based on the ideXlab platform.
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synthesis bioactivity and cloning of the l type calcium channel blocker Omega Conotoxin txvii
Biochemistry, 1999Co-Authors: Toru Sasaki, Mike Fainzilber, Zhongping Feng, Randolph Scott, Nikita Grigoriev, Naweed I Syed, Kazuki SatoAbstract:: Omega-Conotoxin TxVII is the first Conotoxin reported to block L-type currents. In contrast to other Omega-Conotoxins, its sequence is characterized by net negative charge and high hydrophobicity, although it retains the Omega-Conotoxin cysteine framework. In order to obtain structural information and to supply material for further characterization of its biological function, we synthesized TxVII and determined its disulfide bond pairings. Because a linear precursor with free SH groups showed a strong tendency to aggregate and to polymerize, we examined many different conditions for air oxidation and concluded that a mixture of cationic buffer and hydrophobic solvent was the most effective for the folding of TxVII. Synthetic TxVII was shown to suppress the slowly inactivating voltage-dependent calcium current in cultured Lymnaea RPeD1 neurons and furthermore to suppress synaptic transmission between these neurons and their follower cells. In contrast, TxVII did not block calcium flux through L-type channels in PC12 cells, suggesting a phyletic or subtype specificity in this channel family. Disulfide bond pairings of TxVII and its isomers were determined by enzymatic fragmentation in combination with chemical synthesis, thus revealing that TxVII has the same disulfide bond pattern as other Omega-Conotoxins. Furthermore, the CD spectrum of TxVII is similar to those of Omega-Conotoxins MVIIA and MVIIC. The precursor sequence of TxVII was determined by cDNA cloning and shown to be closest to that of delta-Conotoxin TxVIA, a sodium channel inactivation inhibitor. Thus TxVII conserves the structural fold of other Omega-Conotoxins, and the TxVIA/TxVII branch of this family reveals the versatility of its structural scaffold, allowing evolution of structurally related peptides to target different channels.
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three dimensional structure in solution of the calcium channel blocker Omega Conotoxin mviia
Biochemistry, 1995Co-Authors: Toshiyuki Kohno, Kuniko Kobayashi, Yoshio Kodera, Tadakazu Maeda, Kazuki SatoAbstract:: The three-dimensional solution structure of Omega-Conotoxin MVIIA, a 25-mer peptide antagonist of N-type calcium channels, was determined by two-dimensional 1H NMR spectroscopy with simulated annealing calculations. A total of 13 converged structures of Omega-Conotoxin MVIIA were obtained on the basis of 273 experimental constraints, including 232 distance constraints obtained from nuclear Overhauser effect (NOE) connectivities, 22 torsion angle (phi, chi 1) constraints, and 19 constraints associated with hydrogen bonds and disulfide bonds. The atomic root mean square difference about the averaged coordinate positions is 0.47 +/- 0.08 A for the backbone atoms (N, C alpha, C) and 1.27 +/- 0.14 A for all heavy atoms of the entire peptide. The molecular structure of Omega-Conotoxin MVIIA is composed of a short triple-stranded antiparallel beta-sheet. The overall beta-sheet topology is +2x, -1, which is the same as that reported for Omega-Conotoxin GVIA, another N-type calcium channel blocker. The orientation of beta-stranded structure is similar to each other, suggesting that the conserved disulfide bond combination is essential for the molecular folding. We have recently determined by using alanine substitution analyses that Tyr 13 is essential for the activity of both toxins. On the basis of functional and structural analysis, it is shown that both Omega-Conotoxin MVIIA and GVIA retain a similar conformation to locate Tyr 13 in the appropriate position to allow binding to N-type calcium channels. These results provide a molecular basis for understanding the mechanism of calcium channel modulation through the toxin-channel interaction and insight into the discrimination of different subtypes of calcium channels.
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hydroxyl group of tyr13 is essential for the activity of Omega Conotoxin gvia a peptide toxin for n type calcium channel
Journal of Biological Chemistry, 1994Co-Authors: Masami Takahashi, A Ogura, T Kohno, Y Kudo, Kazuki SatoAbstract:Abstract A series of analogs of Omega-Conotoxin GVIA, a peptide neurotoxin having 27 amino acid residues with three disulfide bridges, were synthesized by replacing each amino acid residue except for Cys and Hyp with Ala. CD spectra were virtually identical between native and all of the analogs, indicating the overall conformations were not changed by the substitutions. The inhibitory effects of these analogs on 125I-Omega-Conotoxin GVIA binding to chick brain synaptic plasma membranes showed that replacement of Tyr13 with Ala drastically lowered the affinity of the toxin to the N-type Ca2+ channel. Substitution of Tyr13 with Phe also showed reduction of the affinity, indicating that the hydroxyl group of Tyr13 is critical for binding. Since Lys2 is also important for binding (Sato, K. Park, N.-G., Kohno, T. Maeda, T., Kim, J.-I., Kato, R., and Takahashi, M. (1993) Biochem. Biophys. Res. Commun. 194, 1292-1296), we propose a two-point binding model in which Tyr13 and Lys2 interact with specific amino acid residues of the Ca2+ channel through hydrogen bonding and ionic interaction, respectively.
Ingemar Engberg - One of the best experts on this subject based on the ideXlab platform.
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nifedipine and Omega Conotoxin sensitive ca2 conductances in guinea pig substantia nigra pars compacta neurones
The Journal of Physiology, 1993Co-Authors: Steen Nedergaard, J A Flatman, Ingemar EngbergAbstract:1. The membrane properties of substantia nigra pars compacta (SNc) neurones were recorded in guinea-pig in vitro brain slices. 2. In the presence of tetrodotoxin (TTX) a Ca(2+)-dependent slow oscillatory potential (SOP) was generated. Application of 0.5-20 microM nifedipine abolished both spontaneous and evoked SOPs. A tetraethylammonium chloride (TEA)-promoted high-threshold Ca2+ spike (HTS) was little affected by nifedipine. On the other hand, Omega-Conotoxin applied either locally or via the perfusion medium (1-10 microM) blocked a part of the HTS, but it did not alter the SOP. 3. In normal medium nifedipine blocked the spontaneous discharge, decreased the interspike interval (ISI) recorded during depolarizing current injections and selectively reduced the slow component of the spike after-hyperpolarization (AHP). Omega-Conotoxin decreased both the rising and falling slopes of the normal action potential, reduced the peak amplitude of the spike AHP, and, in some of the neurones, reduced the ISI during depolarization. The Na+ spikes recorded in Ca(2+)-free medium were not altered by Omega-Conotoxin. 4. The SOP was not blocked by octanol (100-200 microM), amiloride (100-250 microM), or Ni2+ (100-300 microM). However, at 500 microM Ni2+ attenuated the SOP. 5. Application of apamin (0.5-2.0 microM) induced irregular firing or bursting, abolished the slow component of the spike AHP and reduced its peak amplitude. In the presence of TTX and apamin long-duration plateau potentials occurred, which were subsequently blocked by nifedipine. 6. In Ca(2+)-free, Co(2+)-containing medium TTX-sensitive spikes and voltage plateaux were generated by depolarizing current pulses. It is suggested that a persistent Na+ conductance underlies the plateaux, which may be co-activated during the SOP. 7. The results suggest that the Ca2+ currents underlying the SOP and the HTS are different and that they activate at least two Ca(2+)-dependent K+ conductances. These conductances play major roles in the maintenance of spontaneous discharge and in control of membrane excitability.
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Nifedipine‐ and Omega‐Conotoxin‐sensitive Ca2+ conductances in guinea‐pig substantia nigra pars compacta neurones.
The Journal of Physiology, 1993Co-Authors: Steen Nedergaard, J A Flatman, Ingemar EngbergAbstract:1. The membrane properties of substantia nigra pars compacta (SNc) neurones were recorded in guinea-pig in vitro brain slices. 2. In the presence of tetrodotoxin (TTX) a Ca(2+)-dependent slow oscillatory potential (SOP) was generated. Application of 0.5-20 microM nifedipine abolished both spontaneous and evoked SOPs. A tetraethylammonium chloride (TEA)-promoted high-threshold Ca2+ spike (HTS) was little affected by nifedipine. On the other hand, Omega-Conotoxin applied either locally or via the perfusion medium (1-10 microM) blocked a part of the HTS, but it did not alter the SOP. 3. In normal medium nifedipine blocked the spontaneous discharge, decreased the interspike interval (ISI) recorded during depolarizing current injections and selectively reduced the slow component of the spike after-hyperpolarization (AHP). Omega-Conotoxin decreased both the rising and falling slopes of the normal action potential, reduced the peak amplitude of the spike AHP, and, in some of the neurones, reduced the ISI during depolarization. The Na+ spikes recorded in Ca(2+)-free medium were not altered by Omega-Conotoxin. 4. The SOP was not blocked by octanol (100-200 microM), amiloride (100-250 microM), or Ni2+ (100-300 microM). However, at 500 microM Ni2+ attenuated the SOP. 5. Application of apamin (0.5-2.0 microM) induced irregular firing or bursting, abolished the slow component of the spike AHP and reduced its peak amplitude. In the presence of TTX and apamin long-duration plateau potentials occurred, which were subsequently blocked by nifedipine. 6. In Ca(2+)-free, Co(2+)-containing medium TTX-sensitive spikes and voltage plateaux were generated by depolarizing current pulses. It is suggested that a persistent Na+ conductance underlies the plateaux, which may be co-activated during the SOP. 7. The results suggest that the Ca2+ currents underlying the SOP and the HTS are different and that they activate at least two Ca(2+)-dependent K+ conductances. These conductances play major roles in the maintenance of spontaneous discharge and in control of membrane excitability.
Steen Nedergaard - One of the best experts on this subject based on the ideXlab platform.
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nifedipine and Omega Conotoxin sensitive ca2 conductances in guinea pig substantia nigra pars compacta neurones
The Journal of Physiology, 1993Co-Authors: Steen Nedergaard, J A Flatman, Ingemar EngbergAbstract:1. The membrane properties of substantia nigra pars compacta (SNc) neurones were recorded in guinea-pig in vitro brain slices. 2. In the presence of tetrodotoxin (TTX) a Ca(2+)-dependent slow oscillatory potential (SOP) was generated. Application of 0.5-20 microM nifedipine abolished both spontaneous and evoked SOPs. A tetraethylammonium chloride (TEA)-promoted high-threshold Ca2+ spike (HTS) was little affected by nifedipine. On the other hand, Omega-Conotoxin applied either locally or via the perfusion medium (1-10 microM) blocked a part of the HTS, but it did not alter the SOP. 3. In normal medium nifedipine blocked the spontaneous discharge, decreased the interspike interval (ISI) recorded during depolarizing current injections and selectively reduced the slow component of the spike after-hyperpolarization (AHP). Omega-Conotoxin decreased both the rising and falling slopes of the normal action potential, reduced the peak amplitude of the spike AHP, and, in some of the neurones, reduced the ISI during depolarization. The Na+ spikes recorded in Ca(2+)-free medium were not altered by Omega-Conotoxin. 4. The SOP was not blocked by octanol (100-200 microM), amiloride (100-250 microM), or Ni2+ (100-300 microM). However, at 500 microM Ni2+ attenuated the SOP. 5. Application of apamin (0.5-2.0 microM) induced irregular firing or bursting, abolished the slow component of the spike AHP and reduced its peak amplitude. In the presence of TTX and apamin long-duration plateau potentials occurred, which were subsequently blocked by nifedipine. 6. In Ca(2+)-free, Co(2+)-containing medium TTX-sensitive spikes and voltage plateaux were generated by depolarizing current pulses. It is suggested that a persistent Na+ conductance underlies the plateaux, which may be co-activated during the SOP. 7. The results suggest that the Ca2+ currents underlying the SOP and the HTS are different and that they activate at least two Ca(2+)-dependent K+ conductances. These conductances play major roles in the maintenance of spontaneous discharge and in control of membrane excitability.
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Nifedipine‐ and Omega‐Conotoxin‐sensitive Ca2+ conductances in guinea‐pig substantia nigra pars compacta neurones.
The Journal of Physiology, 1993Co-Authors: Steen Nedergaard, J A Flatman, Ingemar EngbergAbstract:1. The membrane properties of substantia nigra pars compacta (SNc) neurones were recorded in guinea-pig in vitro brain slices. 2. In the presence of tetrodotoxin (TTX) a Ca(2+)-dependent slow oscillatory potential (SOP) was generated. Application of 0.5-20 microM nifedipine abolished both spontaneous and evoked SOPs. A tetraethylammonium chloride (TEA)-promoted high-threshold Ca2+ spike (HTS) was little affected by nifedipine. On the other hand, Omega-Conotoxin applied either locally or via the perfusion medium (1-10 microM) blocked a part of the HTS, but it did not alter the SOP. 3. In normal medium nifedipine blocked the spontaneous discharge, decreased the interspike interval (ISI) recorded during depolarizing current injections and selectively reduced the slow component of the spike after-hyperpolarization (AHP). Omega-Conotoxin decreased both the rising and falling slopes of the normal action potential, reduced the peak amplitude of the spike AHP, and, in some of the neurones, reduced the ISI during depolarization. The Na+ spikes recorded in Ca(2+)-free medium were not altered by Omega-Conotoxin. 4. The SOP was not blocked by octanol (100-200 microM), amiloride (100-250 microM), or Ni2+ (100-300 microM). However, at 500 microM Ni2+ attenuated the SOP. 5. Application of apamin (0.5-2.0 microM) induced irregular firing or bursting, abolished the slow component of the spike AHP and reduced its peak amplitude. In the presence of TTX and apamin long-duration plateau potentials occurred, which were subsequently blocked by nifedipine. 6. In Ca(2+)-free, Co(2+)-containing medium TTX-sensitive spikes and voltage plateaux were generated by depolarizing current pulses. It is suggested that a persistent Na+ conductance underlies the plateaux, which may be co-activated during the SOP. 7. The results suggest that the Ca2+ currents underlying the SOP and the HTS are different and that they activate at least two Ca(2+)-dependent K+ conductances. These conductances play major roles in the maintenance of spontaneous discharge and in control of membrane excitability.
Nicholas Dale - One of the best experts on this subject based on the ideXlab platform.
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gabab receptors modulate an Omega Conotoxin sensitive calcium current that is required for synaptic transmission in the xenopus embryo spinal cord
The Journal of Neuroscience, 1994Co-Authors: Matthew J Wall, Nicholas DaleAbstract:Activation of GABAB receptors in the Xenopus embryo, a simple vertebrate, causes presynaptic inhibition of transmitter release from glycinergic spinal neurons and an increase in action potential threshold. To investigate the underlying mechanisms of GABAB receptor action, we have made whole-cell voltage-clamp recordings from acutely isolated Xenopus embryo spinal neurons. The GABAB receptor agonist baclofen caused a reversible reduction in the amplitude of Ca2+ currents. This reduction of Ca2+ currents appeared to be voltage dependent as it was removed at very positive potentials. Since the specific GABAB antagonists CGP35348, phaclofen, and 2-hydroxysaclofen all blocked the reduction in Ca2+ currents, we concluded that the modulation of the Ca2+ current was mediated by GABAB receptors. We have investigated the pharmacological identity of the Ca2+ current modulated by baclofen using the selective blocker Omega-Conotoxin, fraction GVIA (Omega-CgTX). Omega-CgTX selectively blocked voltage-gated Ca2+ currents without affecting the voltage-gated Na+ current. Omega-CgTX substantially occluded the action of baclofen, suggesting that GABAB receptors modulate an Omega-CgTX-sensitive Ca2+ current. Since GABAB receptors mediate presynaptic inhibition, we have studied the involvement of the Omega-CgTX-sensitive Ca2+ current in synaptic transmission in the intact spinal cord. Inhibitory interneuron axons were stimulated to evoke monosynaptic IPSPs in motoneurons, and recorded intracellularly. Since Omega-CgTX blocked inhibitory transmission, we concluded that the Omega-CgTX-sensitive Ca2+ current plays an essential role in transmitter release. If modulation of this current were to occur in nerve terminals, it could contribute to the GABAB receptor-mediated presynaptic inhibition of transmitter release.(ABSTRACT TRUNCATED AT 250 WORDS)
Raymond S Norton - One of the best experts on this subject based on the ideXlab platform.
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refined solution structure of Omega Conotoxin gvia implications for calcium channel binding
Journal of Peptide Research, 1999Co-Authors: Paul K Pallaghy, Raymond S NortonAbstract:: The polypeptide Omega-Conotoxin GVIA (GVIA) is an N-type calcium channel blocker from the venom of Conus geographus, a fish-hunting cone shell. Here we describe a high-resolution solution structure of this member of the 'inhibitor cystine knot' protein family. The structure, based on NMR data acquired at 600 MHz, has mean pairwise RMS differences of 0.25 +/- 0.06 and 1.07 +/- 0.14 A over the backbone heavy atoms and all heavy atoms, respectively. The solvent-accessible side chains are better defined than in previously published structures and provide an improved basis for docking GVIA with models of the calcium channel. Moreover, some side chain interactions important in GVIA folding in vitro and in stabilizing the native structure are defined clearly in the refined structure. Two qualitatively different backbone conformations in the segment from Thr11 to Asn14 persisted in the restrained simulated annealing calculations until a small number of lower bound constraints was included to prevent close contacts from occurring that did not correspond with peaks in the NOESY spectrum. It is possible that GVIA is genuinely flexible at this segment, spending a finite time in the alternative conformation, and this may influence its interaction with the calcium channel.
