The Experts below are selected from a list of 30300 Experts worldwide ranked by ideXlab platform
Gerald W. Zamponi - One of the best experts on this subject based on the ideXlab platform.
-
Heterodimerization of ORL1 and Opioid Receptors and Its Consequences for N-Type Calcium Channel Regulation
The Journal of biological chemistry, 2009Co-Authors: Rhian M. Evans, Haitao You, Shahid Hameed, Christophe Altier, Alexandre Mezghrani, Emmanuel Bourinet, Gerald W. ZamponiAbstract:We have investigated the heterodimerization of ORL1 receptors and classical members of the opioid receptor family. All three classes of opioid receptors could be co-immunoprecipitated with ORL1 receptors from both transfected tsA-201 cell lysate and rat dorsal root ganglia lysate, suggesting that these receptors can form heterodimers. Consistent with this hypothesis, in cells expressing either one of the opioid receptors together with ORL1, prolonged ORL1 receptor activation via nociceptin application resulted in internalization of the opioid receptors. Conversely, μ-, δ-, and κ-opioid receptor activation with the appropriate ligands triggered the internalization of ORL1. The μ-opioid receptor/ORL1 receptor heterodimers were shown to associate with N-Type Calcium Channels, with activation of μ-opioid receptors triggering N-Type Channel internalization, but only in the presence of ORL1. Furthermore, the formation of opioid receptor/ORL1 receptor heterodimers attenuated the ORL1 receptor-mediated inhibition of N-Type Channels, in part because of constitutive opioid receptor activity. Collectively, our data support the existence of heterodimers between ORL1 and classical opioid receptors, with profound implications for effectors such as N-Type Calcium Channels.
-
Scaffold-based design and synthesis of potent N-Type Calcium Channel blockers.
Bioorganic & medicinal chemistry letters, 2009Co-Authors: Gerald W. Zamponi, Hassan Pajouhesh, Zhong Ping Feng, Lingyun Zhang, Yanbing Ding, Francesco Belardetti, Hassan Pajouhesh, David Dolphin, Lester A. Mitscher, Terrance P. SnutchAbstract:The therapeutic agents flunarizine and lomerizine exhibit inhibitory activities against a variety of ion Channels and neurotransmitter receptors. We have optimized their scaffolds to obtain more selective N-Type Calcium Channel blockers. During this optimization, we discovered NP118809 and NP078585, two potent N-Type Calcium Channel blockers which have good selectivity over L-type Calcium Channels. Upon intraperitoneal administration both compounds exhibit analgesic activity in a rodent model of inflammatory pain. NP118809 further exhibits a number of favorable preclinical characteristics as they relate to overall pharmacokinetics and minimal off-target activity including the hERG potassium Channel.
-
Scanning mutagenesis reveals a role for serine 189 of the heterotrimeric G-protein beta 1 subunit in the inhibition of N-Type Calcium Channels.
Journal of neurophysiology, 2006Co-Authors: H. William Tedford, Alexandra E. Kisilevsky, Jean B. Peloquin, Gerald W. ZamponiAbstract:Direct interactions between the presynaptic N-Type Calcium Channel and the β subunit of the heterotrimeric G-protein complex cause voltage-dependent inhibition of N-Type Channel activity, crucially influencing neurotransmitter release and contributing to analgesia caused by opioid drugs. Previous work using chimeras of the G-protein β subtypes Gβ1 and Gβ5 identified two 20–amino acid stretches of structurally contiguous residues on the Gβ1 subunit as critical for inhibition of the N-Type Channel. To identify key modulation determinants within these two structural regions, we performed scanning mutagenesis in which individual residues of the Gβ1 subunit were replaced by corresponding Gβ5 residues. Our results show that Gβ1 residue Ser189 is critical for N-Type Calcium Channel modulation, whereas none of the other Gβ1 mutations caused statistically significant effects on the ability of Gβ1 to inhibit N-Type Channels. Structural modeling shows residue 189 is surface exposed, consistent with the idea that it ...
-
Several structural domains contribute to the regulation of N-Type Calcium Channel inactivation by the beta 3 subunit.
The Journal of biological chemistry, 2003Co-Authors: Stephanie C. Stotz, Wendy Barr, John E. Mcrory, Lina Chen, Scott E. Jarvis, Gerald W. ZamponiAbstract:Abstract Calcium Channel β subunits are essential regulatory elements of the gating properties of high voltage-activated Calcium Channels. Co-expression with β3 subunits typically accelerates inactivation, whereas co-expression with β4 subunits results in a slowly inactivating phenotype. Here, we have examined the molecular basis of the differential effect of these two subunits on the inactivation characteristics of Cav2.2 + α2-δ1 N-Type Calcium Channels by creating a series of 22 chimeric β subunits that are based on various combinations of variable and conserved regions of the parent β subunit isoforms. Our data show that replacement of the N terminus region of β4 with a corresponding 14-amino acid stretch of β3 sequence accelerates the inactivation kinetics to levels seen with wild type β3. A similar kinetic speeding is observed by a concomitant substitution of the second conserved and variable regions, but not when these regions are substituted individually, suggesting that 1) the second variable and conserved regions cooperatively regulate N-Type Calcium Channel inactivation and 2) that there are two redundant mechanisms that allow the β3 subunit to accelerate N-Type Channel inactivation. In contrast with previous reports in Cav2.1 Calcium Channels, deletion of the C-terminal region of Cav2.2 did not alter the regulation of the Channel by wild type and chimeric β subunits. Hence, the molecular underpinnings of β subunit regulation of voltage-gated Calcium Channels appear to vary with Calcium Channel subtype.
-
Molecular determinants of syntaxin 1 modulation of N-Type Calcium Channels.
The Journal of biological chemistry, 2002Co-Authors: Scott E. Jarvis, Zhong Ping Feng, Wendy Barr, Jawed Hamid, Gerald W. ZamponiAbstract:We have previously reported that syntaxin 1A, a component of the presynaptic SNARE complex, directly modulates N-Type Calcium Channel gating in addition to promoting tonic G-protein inhibition of the Channels, whereas syntaxin 1B affects Channel gating but does not support G-protein modulation (Jarvis, S. E., and Zamponi, G. W. (2001) J. Neurosci. 21, 2939-2948). Here, we have investigated the molecular determinants that govern the action of syntaxin 1 isoforms on N-Type Calcium Channel function. In vitro evidence shows that both syntaxin 1 isoforms physically interact with the G-protein beta subunit and the synaptic protein interaction (synprint) site contained within the N-Type Calcium Channel domain II-III linker region. Moreover, in vitro evidence suggests that distinct domains of syntaxin participate in each interaction, with the COOH-terminal SNARE domain (residues 183-230) binding to Gbeta and the N-terminal (residues 1-69) binding to the synprint motif of the Channel. Electrophysiological analysis of chimeric syntaxin 1A/1B constructs reveals that the variable NH(2)-terminal domains of syntaxin 1 are responsible for the differential effects of syntaxin 1A and 1B on N-Type Calcium Channel function. Because syntaxin 1 exists in both "open" and "closed" conformations during exocytosis, we produced a constitutively open form of syntaxin 1A and found that it still promoted G-protein inhibition of the Channels, but it did not affect N-Type Channel availability. This state dependence of the ability of syntaxin 1 to mediate N-Type Calcium Channel availability suggests that syntaxin 1 dynamically regulates N-Type Channel function during various steps of exocytosis. Finally, syntaxin 1A appeared to compete with Ggamma for the Gbeta subunit both in vitro and under physiological conditions, suggesting that syntaxin 1A may contain a G-protein gamma subunit-like domain.
Masami Takahashi - One of the best experts on this subject based on the ideXlab platform.
-
Tyr13 Is Essential for the Activity of ω-Conotoxin MVIIA and GVIA, Specific N-Type Calcium Channel Blockers
Biochemical and biophysical research communications, 1995Co-Authors: Jae Il Kim, Masami Takahashi, Atsuko Ohtake, A. Wakamiya, Kazuki SatoAbstract:Abstract Two analogs of ω-conotoxin MVIIA, a 25mer peptide neurotoxin, were synthesized by replacing Lys2 or Tyr13 with Ala. The activities of synthetic analogs were estimated from the inhibitory action on 125I-ω-conotoxin GVIA binding to chick brain synaptic plasma membranes. As in the case of ω-conotoxin GVIA, replacement of Tyr13 resulted in an enormous reduction in activity. In contrast, substitution of Ala for Lys2 gave only a small effect. These results indicate that Tyr13 is a critical amino acid of ω-conotoxin MVIIA and GVIA for blocking N-Type Calcium Channel function.
-
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. KudoAbstract: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.
-
Purification of the N-Type Calcium Channel associated with syntaxin and synaptotagmin. A complex implicated in synaptic vesicle exocytosis.
The Journal of biological chemistry, 1994Co-Authors: Christian Lévêque, Masami Takahashi, Kazuki Sato, O. El Far, Nicole Martin-moutot, Rika Kato, Michael SeagarAbstract:Abstract omega-Conotoxin-sensitive N-Type Calcium Channels control neurotransmitter release at the nerve terminal and interact with proteins implicated in secretion. Solubilized omega-conotoxin receptors from rat brain synaptic membrane were immunoprecipitated by antibodies against Calcium Channel alpha 1 subunits, syntaxin, and a 105-kDa plasma membrane protein. A multimeric complex, composed of Calcium Channel subunits, and synaptic proteins that showed varying degrees of association, was purified by a procedure involving anti-syntaxin immunoaffinity chromatography. A 250-kDa N-Type alpha 1 subunit, containing cAMP-dependent phosphorylation site(s), was identified by photoaffinity labeling with 125I-azidonitrobenzoyl omega-conotoxin and immunoblotting with sequence-directed antibodies. An immunologically related 210-kDa form of the alpha 1 subunit was detected that displayed different pharmacological and regulatory properties. Protein bands of 140, 70, 58, and 35 kDa comigrated with purified alpha 1 subunits upon sucrose gradient centrifugation, whereas the 105-kDa protein was removed. The 58- and 35-kDa bands contained, respectively, the synaptic vesicle protein synaptotagmin and syntaxin, a plasma membrane protein that binds synaptic vesicle proteins. Purified omega-contoxin receptors were quantitatively immunoprecipitated by anti-syntaxin antibodies. These proteins may constitute an isolated exocytotic complex in which the N-Type Calcium Channel tightly interacts with a synaptic vesicle docking site.
-
hpc 1 is associated with synaptotagmin and omega conotoxin receptor
Journal of Biological Chemistry, 1992Co-Authors: A Yoshida, A Omori, R Kuwahara, Masami TakahashiAbstract:Abstract Monoclonal antibodies were produced that recognize a membrane protein of 35,000 Da (p35) expressed in brain and adrenal medulla. They immunoprecipitated 50% of omega-conotoxin (omega-CgTX) receptor, a putative N-Type Calcium Channel, solubilized from rat brain. Anti-synaptotagmin (p65) antibodies also immunoprecipitate omega-CgTX receptor (Leveque, C., Hoshino, T., David, P., Shoji-Kasai, Y., Leys, K., Omori, A., Lang, B., El Far, O., Sato, K., Martin-Moutot, N., Newsom-Davis, J., Takahashi, M., and Seagar, M.J. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 3625-3629); however, immunoprecipitation by anti-p35 antibodies and anti-synaptotagmin antibodies was not additive. Furthermore, both p35 and synaptotagmin were recovered in the immunoprecipitates with anti-synaptotagmin and anti-p35 antibodies, respectively, indicating that a population of omega-CgTX receptor exists as a ternary complex with synaptotagmin and p35. A cDNA coding p35 was isolated from a rat brain cDNA library by immuno-screening, and the primary structure of the protein was revealed to be identical to that of HPC-1 (Inoue, A., Obata, K., and Akagawa, K. (1992) J. Biol. Chem. 267, 10613-10619). HPC-1 has a putative transmembrane segment at the C terminus and four heptad motifs, which may be involved in protein-protein interaction. These results suggest that HPC-1 may play a role in neurotransmitter release from nerve terminals by associating with omega-CgTX-sensitive N-Type Calcium Channel and synaptotagmin.
Fawu Dong - One of the best experts on this subject based on the ideXlab platform.
-
valepotriates from the roots and rhizomes of valeriana jatamansi jones as novel n type Calcium Channel antagonists
Frontiers in Pharmacology, 2018Co-Authors: Fawu Dong, Hehai Jiang, Liu Yang, Ye Gong, Dan Yang, Jian Yang, Yin NianAbstract:The roots and rhizomes of V. jatamansi have long been used as folk medicine in Asia and usually named as ″Zhizhuxiang″ in Chinese for the treatment of abdominal distention and pain. However, its active ingredients and molecular targets for treatment of abdominal pain remain unrevealed. Inhibitors of Cav2.2 N-Type voltage-gated Calcium Channels (VGCCs) are actively sought after for their potential in treating pain, especially chronic pain. As far as we know, the method used for seeking analgesic active ingredient from plant material has rarely been reported. The analgesic potentials of the EtOH extract (0.01 mg/ml) of the roots and rhizomes of V. jatamansi and its EtOAc, n-BuOH and H2O soluble parts (0.01 mg/ml, respectively) were tested herein on Cav2.2, using whole-oocyte recordings in vitro by tow-electrode voltage clamp. The results indicated that the EtOAc-soluble part exhibiting the most potent inhibition of Cav2.2 peak current (20 mv). The EtOAc-soluble part was then subjected to silica gel column chromatography (CC) and giving 9 fractions. Phytochemical studies were carried out by repeated CC and extensive spectroscopic analysis after the fraction was identified active and got seventeen compounds (1-17). All isolates were then sent further for bioactive verification (1 and 3 at concentration of 10 μM, others at 30 μM). In addition, the selectivity of compounds 1 and 3 were tested on various ion Channels including Cav1.2, Cav2.1 and Cav3.1 VGCCs and Kv1.2, Kv2.1, Kv3.1 and BK potassium Channels. The results indicated that compounds 1 and 3 (an abundant compound) inhibited Cav2.2 with an EC50 of 3.3 μM and 4.8 μM respectively and had weaker or no effect on Cav1.2, Cav2.1 and Cav3.1 VGCCs and Kv1.2, Kv2.1, Kv3.1 and BK potassium Channels. Compounds 1 and 3 appear to act as allosteric modulators rather than pore blockers of Cav2.2, which may play crucial role in attenuating nociception. The results of present research indicated that the ethnopharmacological utilization of V. jatamansi for relieving the abdominal distention and pain may mediate through Cav2.2 Channel. Our work is the first demonstration of inhibition of Cav2.2 by iridoids, which may provide a fresh source for finding new analgesics.
-
Valepotriates From the Roots and Rhizomes of Valeriana jatamansi Jones as Novel N-Type Calcium Channel Antagonists
Frontiers Media S.A., 2018Co-Authors: Fawu Dong, Hehai Jiang, Liu Yang, Ye Gong, Dan YangAbstract:The roots and rhizomes of Valeriana jatamansi have long been used as folk medicine in Asia and usually named as “Zhizhuxiang” in Chinese for the treatment of abdominal distention and pain. However, its active ingredients and molecular targets for treatment of abdominal pain remain unrevealed. Inhibitors of Cav2.2 N-Type voltage-gated Calcium Channels (VGCCs) are actively sought after for their potential in treating pain, especially chronic pain. As far as we know, the method used for seeking analgesic active ingredient from plant material has rarely been reported. The analgesic potentials of the EtOH extract (0.01 mg/ml) of the roots and rhizomes of V. jatamansi and its EtOAc, n-BuOH and H2O soluble parts (0.01 mg/ml, respectively) were tested herein on Cav2.2, using whole-oocyte recordings in vitro by tow-electrode voltage clamp. The results indicated that the EtOAc-soluble part exhibited the most potent inhibition of Cav2.2 peak current (20 mv). The EtOAc-soluble part was then subjected to silica gel column chromatography (CC) and giving 9 fractions. Phytochemical studies were carried out by repeated CC and extensive spectroscopic analyses after the fraction (0.01 mg/ml) was identified to be active and got seventeen compounds (1–17). All isolates were then sent for further bioactive verification (1 and 3 at concentration of 10 μM, others at 30 μM). In addition, the selectivity of the active compounds 1 and 3 were tested on various ion Channels including Cav1.2, Cav2.1 and Cav3.1 VGCCs and Kv1.2, Kv2.1, Kv3.1 and BK potassium Channels. The results indicated that compound 1 and 3 (an abundant compound) inhibited Cav2.2 with an EC50 of 3.3 and 4.8 μM, respectively, and had weaker or no effect on Cav1.2, Cav2.1 and Cav3.1 VGCCs and Kv1.2, Kv2.1, Kv3.1 and BK potassium Channels. Compounds 1 and 3 appear to act as allosteric modulators rather than pore blockers of Cav2.2, which may play crucial role in attenuating nociception. The results of present research indicated that the ethnopharmacological utilization of V. jatamansi for relieving the abdominal distention and pain may mediate through Cav2.2 Channel. Our work is the first demonstration of inhibition of Cav2.2 by iridoids, which may provide a fresh source for finding new analgesics
Michael Seagar - One of the best experts on this subject based on the ideXlab platform.
-
Purification of the N-Type Calcium Channel associated with syntaxin and synaptotagmin. A complex implicated in synaptic vesicle exocytosis.
The Journal of biological chemistry, 1994Co-Authors: Christian Lévêque, Masami Takahashi, Kazuki Sato, O. El Far, Nicole Martin-moutot, Rika Kato, Michael SeagarAbstract:Abstract omega-Conotoxin-sensitive N-Type Calcium Channels control neurotransmitter release at the nerve terminal and interact with proteins implicated in secretion. Solubilized omega-conotoxin receptors from rat brain synaptic membrane were immunoprecipitated by antibodies against Calcium Channel alpha 1 subunits, syntaxin, and a 105-kDa plasma membrane protein. A multimeric complex, composed of Calcium Channel subunits, and synaptic proteins that showed varying degrees of association, was purified by a procedure involving anti-syntaxin immunoaffinity chromatography. A 250-kDa N-Type alpha 1 subunit, containing cAMP-dependent phosphorylation site(s), was identified by photoaffinity labeling with 125I-azidonitrobenzoyl omega-conotoxin and immunoblotting with sequence-directed antibodies. An immunologically related 210-kDa form of the alpha 1 subunit was detected that displayed different pharmacological and regulatory properties. Protein bands of 140, 70, 58, and 35 kDa comigrated with purified alpha 1 subunits upon sucrose gradient centrifugation, whereas the 105-kDa protein was removed. The 58- and 35-kDa bands contained, respectively, the synaptic vesicle protein synaptotagmin and syntaxin, a plasma membrane protein that binds synaptic vesicle proteins. Purified omega-contoxin receptors were quantitatively immunoprecipitated by anti-syntaxin antibodies. These proteins may constitute an isolated exocytotic complex in which the N-Type Calcium Channel tightly interacts with a synaptic vesicle docking site.
Kazuki Sato - One of the best experts on this subject based on the ideXlab platform.
-
Tyr13 Is Essential for the Activity of ω-Conotoxin MVIIA and GVIA, Specific N-Type Calcium Channel Blockers
Biochemical and biophysical research communications, 1995Co-Authors: Jae Il Kim, Masami Takahashi, Atsuko Ohtake, A. Wakamiya, Kazuki SatoAbstract:Abstract Two analogs of ω-conotoxin MVIIA, a 25mer peptide neurotoxin, were synthesized by replacing Lys2 or Tyr13 with Ala. The activities of synthetic analogs were estimated from the inhibitory action on 125I-ω-conotoxin GVIA binding to chick brain synaptic plasma membranes. As in the case of ω-conotoxin GVIA, replacement of Tyr13 resulted in an enormous reduction in activity. In contrast, substitution of Ala for Lys2 gave only a small effect. These results indicate that Tyr13 is a critical amino acid of ω-conotoxin MVIIA and GVIA for blocking N-Type Calcium Channel function.
-
Purification of the N-Type Calcium Channel associated with syntaxin and synaptotagmin. A complex implicated in synaptic vesicle exocytosis.
The Journal of biological chemistry, 1994Co-Authors: Christian Lévêque, Masami Takahashi, Kazuki Sato, O. El Far, Nicole Martin-moutot, Rika Kato, Michael SeagarAbstract:Abstract omega-Conotoxin-sensitive N-Type Calcium Channels control neurotransmitter release at the nerve terminal and interact with proteins implicated in secretion. Solubilized omega-conotoxin receptors from rat brain synaptic membrane were immunoprecipitated by antibodies against Calcium Channel alpha 1 subunits, syntaxin, and a 105-kDa plasma membrane protein. A multimeric complex, composed of Calcium Channel subunits, and synaptic proteins that showed varying degrees of association, was purified by a procedure involving anti-syntaxin immunoaffinity chromatography. A 250-kDa N-Type alpha 1 subunit, containing cAMP-dependent phosphorylation site(s), was identified by photoaffinity labeling with 125I-azidonitrobenzoyl omega-conotoxin and immunoblotting with sequence-directed antibodies. An immunologically related 210-kDa form of the alpha 1 subunit was detected that displayed different pharmacological and regulatory properties. Protein bands of 140, 70, 58, and 35 kDa comigrated with purified alpha 1 subunits upon sucrose gradient centrifugation, whereas the 105-kDa protein was removed. The 58- and 35-kDa bands contained, respectively, the synaptic vesicle protein synaptotagmin and syntaxin, a plasma membrane protein that binds synaptic vesicle proteins. Purified omega-contoxin receptors were quantitatively immunoprecipitated by anti-syntaxin antibodies. These proteins may constitute an isolated exocytotic complex in which the N-Type Calcium Channel tightly interacts with a synaptic vesicle docking site.
