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Hartmut Glossmann - One of the best experts on this subject based on the ideXlab platform.

  • identification of a 27 kda high affinity Phenylalkylamine binding polypeptide as the sigma 1 binding site by photoaffinity labeling and ligand directed antibodies
    Molecular Pharmacology, 1993
    Co-Authors: Fabian F Moebius, J. Striessnig, Gregory G Burrows, Markus Hanner, Eduard Schmid, Hartmut Glossmann
    Abstract:

    The verapamil-like arylazide (-)-[3H]azidopamil specifically photoaffinity labeled two low molecular mass polypeptides, with apparent molecular masses of 22 and 27 kDa, in the endoplasmic reticulum of guinea pig liver, kidney, adrenal gland, and lung. It was recently shown that the 22-kDa polypeptide binds the anti-ischemic Phenylalkylamine (-)-[3H]emopamil and other anti-ischemic drugs with high affinity. We now provide evidence that the photolabeling of the 27-kDa polypeptide is blocked by nanomolar concentrations of sigma ligands [order of potency, haloperidol > pentazocine > 1,3-ditolylguanidine > dextromethorphan > (+)-SKF10,047]. The apparent affinities of these and other drugs closely corresponded to those for 1,3-[3H]ditolylguanidine-labeled sigma binding sites. Based on its high affinity for the (+)-enantiomer [but not the (-)-enantiomer] of SKF10,047 (Ki = 51 nM), pentazocine (Ki = 3 nM), and dextromethorphan (Ki = 30 nM), the (-)-[3H]azidopamil-labeled site on the 27-kDa polypeptide was classified as being of the sigma 1 subtype. Using antiPhenylalkylamine antibodies, we developed a novel immunological detection method that allows the rapid and sensitive staining of the photolabeled 27-kDa polypeptide after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We conclude that the Phenylalkylamines emopamil and azidopamil represent a novel class of sigma ligands, highly suitable for the further structural characterization of polypeptides carrying sigma 1 binding sites.

  • a unique fluorescent Phenylalkylamine probe for l type ca2 channels coupling of Phenylalkylamine receptors to ca2 and dihydropyridine binding sites
    Journal of Biological Chemistry, 1992
    Co-Authors: Hansgunther Knaus, T Moshammer, Hee Chol Kang, Richard P Haugland, Hartmut Glossmann
    Abstract:

    Abstract The first fluorescently labeled Phenylalkylamine, DMBODIPY-PAA (5-(3-[3-(4,4-difluoro-5,7-dimethyl-3a, 4a-diaza-4-bora-indacen-3-yl)propionamido] phenethyl-N-methylamino)-2-isopropyl-2-(3,4,5-trimethoxyphenyl)-valer onitrile) has been introduced for L-type Ca2+ channel research. DMBODIPY-PAA binds reversibly to L-type Ca2+ channels purified from rabbit skeletal muscle microsomes by wheat germ agglutinin-Sepharose chromatography. In this preparation DMBODIPY-PAA labels 412 pmol of Phenylalkylamine receptors/mg of protein with a Kd of 6.82 nM and a favorable signal-to-noise ratio. Therefore DMBODIPY-PAA has a higher affinity for purified Ca2+ channels than the commonly employed radioligands and consequently has assisted in channel purification after prelabeling by simply monitoring receptor-bound fluorescence. (+)-PN200-110 (which is stimulatory for (-)-[3H]desmethoxyverapamil binding to purified Ca2+ channels) inhibits DMBODIPY-PAA labeling. Since these drug interactions are reciprocal, the Phenylalkylamine and dihydropyridine binding sites of the alpha 1-subunit are tightly coupled. Kinetic and equilibrium binding studies with (-)-[3H]desmethoxyverapamil and DMBODIPY-PAA show that Phenylalkylamine binding to L-type Ca2+ channels is dependent on Ca2+. Chelation of divalent metal ions converts Phenylalkylamine receptors into a very low affinity state. This conversion is temperature- and time-dependent and completely reversible (K0.5 for free Ca2+ = 58 nM). This study demonstrates the utility of fluorescent ligands for binding studies with L-type Ca2+ channels and provides evidence for coupling between Ca2+ binding sites and Phenylalkylamine receptors.

  • Novel sites for Phenylalkylamines: characterisation of a sodium-sensitive drug receptor with (-)-[3H]emopamil.
    European Journal of Pharmacology, 1991
    Co-Authors: Christian Zech, R. Staudinger, Johannes Mühlbacher, Hartmut Glossmann
    Abstract:

    Abstract (−)-Emopamil ((S)-emopamil, (2S)-2-isopropyl-5-(methylphenethylamino)-2-phenylvaleronitrile hydrochloride) is a Ca 2+ , antagonistic Phenylalkylamine which also blocks serotonin (5-HT 2 ) receptors and has antiischemic properties. The (-)-[ 3 H]emopamil tissue distribution profile of specific binding is in striking contrast to that observed for (+)-[ 3 H]PN 200-110 or (−)-[ 3 H]desmethoxyverapamil. (−)-[ 3 H]emopamil labels membrane fractions from guinea-pig liver ⪢ adrenal gland > ? lung ≈ ductus deferens ≈ brain ≈ skeletal muscle. Binding to liver membranes was saturable (K D = 12.8 nM, B max = 35 pmol/mg of protein), stereoselective, reversible (K −1 = 0.22 min −1 at 25°C) and inhibited by tetraethylammonium (IC 50 : 1.8 mM) > Li + IC 50 : 12.5 mM) ≈ Na + (IC 50 : 13.6 mM) and [NH 4 + ] (IC 50 : 79.3 mM) but not by Rb + , Cs + or K + . The high-affinity liver membrane binding sites have a pharmacological profile that is distinct from the Phenylalkylamine receptor domain of the voltage-dependent L-type Ca 2+ channel. Similar sites exist in brain and other tissues, albeit with a lower density. Amiodarone, butoprozine and amiloride derivatives bind with high affinity whereas 1,4-dihydropyridines do not interact at all. It is suggested that the novel Phenylalkylamine site is linked to a sodium-dependent carrier or transport system.

  • Very high affinity interaction of DPI 201-106 and BDF 8784 enantiomers with the Phenylalkylamine-sensitive Ca2+-channel in Drosophila head membranes
    British Journal of Pharmacology, 1991
    Co-Authors: Hartmut Glossmann, R. Staudinger, J. Striessnig, R. Greenberg, Christian Zech, L Hall, B. I. Armah
    Abstract:

    1. Piperazinylindoles (DPI 201-106, BDF 8784), drugs known to act on voltage-dependent Na(+)-channels, bind with very high affinity to a Ca2(+)-channel-associated Phenylalkylamine receptor in Drosophila melanogaster head membranes. These compounds and (+)-tetrandrine, a naturally occurring Ca2(+)-antagonist, were the most selective inhibitors for Phenylalkylamine-labelled Drosophila Ca2(+)-channels compared to mammalian L-type Ca2(+)-channels. 2. Replacement of the cyano group by a methyl group in (+)-DPI 201-106 ((+)-BDF 8784) increases the IC50 value for inhibition of Phenylalkylamine labelling of Drosophila Ca2(+)-channels from 0.29 to 2.1 nM but decreases the IC50 value for inhibition of Phenylalkylamine labelling of mammalian skeletal muscle Ca2(+)-channels from 3480 to 49 nM. 3. DPI 201-106 enantiomers completely block (at 0.1 microM) Phenylalkylamine photolabelling of a 136 K polypeptide in Drosophila head membranes whereas 10 microM aconitine or lidocaine are without effect. 4. Assessment of the Ca2(+)-antagonist effects of the substituted DPI 201-106 enantiomers in K(+)-depolarized taenia strips from guinea-pig caecum yielded pA2 values of 6.33 +/- 0.07 for (-)-BDF 8784 and 6.99 +/- 0.17 for (+)-BDF 8784, respectively. 5. Piperazinylindoles, previously believed to act nonspecifically on voltage-dependent mammalian L-type Ca2(+)-channels, therefore have stereoselectivity for a novel binding site and chemical selectivity unrelated to local anaesthetic activity. 6. It is proposed that a very high affinity piperazinylindole-selective site is coupled to the Phenylalkylamine receptor of Drosophila Ca2(+)-channels. These sites are still present on mammalian L-type Ca2(+)-channels but have lower affinity and/or are less tightly coupled to Phenylalkylamine receptors on the alpha 1-subunit.

  • identification of a Phenylalkylamine binding region within the alpha 1 subunit of skeletal muscle ca2 channels
    Proceedings of the National Academy of Sciences of the United States of America, 1990
    Co-Authors: J. Striessnig, Hartmut Glossmann, William A Catterall
    Abstract:

    Abstract The alpha 1 subunit of the skeletal muscle Ca2+ channel has been specifically photoaffinity labeled with the Phenylalkylamine-receptor-selective verapamil derivative (-)-5-(3-azidophenethyl[N-methyl-3H]methylamino)-2-(3,4,5- trimethoxyphenyl)-2-isopropylvaleronitrile ([N-methyl-3H]LU49888). Proteolytic fragments generated by various endoproteases were probed by immunoprecipitation with several sequence-specific antibodies to determine the site of labeling within the primary structure of alpha 1. These results restrict the site of photolabeling by [N-methyl-3H]LU49888 to the region between Glu-1349 and Trp-1391. This segment of alpha 1 contains transmembrane helix S6 of domain IV and the beginning of the long intracellular C-terminal tail. Because of the Phenylalkylamine receptor site is only accessible from the intracellular side of the Ca2+ channel, we propose that the intracellular end of helix IVS6 and the adjacent intracellular amino acid residues play an essential role in formation of the Phenylalkylamine receptor site. The action of the Phenylalkylamines as open-channel blockers suggests that this region may also contribute to formation of the intracellular opening of the transmembrane pore of the Ca2+ channel.

William A Catterall - One of the best experts on this subject based on the ideXlab platform.

  • molecular determinants of high affinity Phenylalkylamine block of l type calcium channels in transmembrane segment iiis6 and the pore region of the α1subunit
    Journal of Biological Chemistry, 1997
    Co-Authors: Gregory H. Hockerman, Barry D. Johnson, Todd Scheuer, Michael R Abbott, William A Catterall
    Abstract:

    Abstract Recent studies of the Phenylalkylamine binding site in the α1C subunit of l-type Ca2+ channels have revealed three amino acid residues in transmembrane segment IVS6 that are critical for high affinity block and are unique to l-type channels. We have extended this analysis of the Phenylalkylamine binding site to amino acid residues in transmembrane segment IIIS6 and the pore region. Twenty-two consecutive amino acid residues in segment IIIS6 were mutated to alanine and the conserved Glu residues in the pore region of each homologous domain were mutated to Gln. Mutant channels were expressed in tsA-201 cells along with the β1b and α2δ auxiliary subunits. Assay for block of Ba2+ current by (−)-D888 at −60 mV revealed that mutation of five amino acid residues in segment IIIS6 and the pore region that are conserved between l-type and non-l-type channels (Tyr1152, Phe1164, Val1165, Glu1118, and Glu1419) and one l-type-specific amino acid (Ile1153) decreased affinity for (−)-D888 from 10–20-fold. Combination of the four mutations in segment IIIS6 increased the IC50 for block by (−)-D888 to approximately 9 μm, similar to the affinity of non-l-type Ca2+ channels for this drug. These results indicate that there are important determinants of Phenylalkylamine binding in both the S6 segments and the pore regions of domains III and IV, some of which are conserved across the different classes of voltage-gated Ca2+ channels. A model of the Phenylalkylamine receptor site at the interface between domains III and IV of the α1 subunit is presented.

  • Distinct effects of mutations in transmembrane segment IVS6 on block of L-type calcium channels by structurally similar Phenylalkylamines.
    Molecular Pharmacology, 1996
    Co-Authors: Barry D. Johnson, Gregory H. Hockerman, Todd Scheuer, William A Catterall
    Abstract:

    The Phenylalkylamines (-)-D888, verapamil, and D600, cause voltage- and use-dependent block of L-type Ca2+ channels and differ from each other only in the number of methoxy groups on each of their two terminal phenyl rings. To study the effects of mutations in the Phenylalkylamine receptor site on block by these drugs, wild-type and mutant Ca2+ channels were transiently expressed in the tsA-201 clone of human embryonic kidney 293 cells. The combined mutations Y1463A, A1467S, and I1470A (mutant YAI) in transmembrane segment S6 of domain IV of the alpha 1c subunit disrupted block by all three Phenylalkylamines. Surprisingly, although this mutation reduced both resting block at -60 mV and depolarized block at +10 mV by (-)-D888, resting and depolarized block by verapamil and D600 were relatively unaffected. In contrast, for all three drugs, use-dependent block during repetitive stimulations was sharply reduced, and the rate of recovery from depolarized block was accelerated for YAI channels. Thus, the effects of the YAI mutation on apparent affinity were specific to (-)-D888, whereas effects on the kinetics of block were observed for all three drugs. Additional experiments with substitution of phenylalanine for Y1463 suggested that (-)-D888 affinity is specifically sensitive to removal of the hydroxyl group of Y1463, whereas effects on the kinetics of block by all three Phenylalkylamines require larger molecular changes, perhaps related to residue size and hydrophobicity. Analysis of the data using a state-dependent model of drug block suggests that these kinetic differences are caused by both changes in drug access to the receptor site and affinity for binding to the inactivated state of the channel. The different effects of the YAI mutations on the actions of (-)-D888, verapamil, and D600 indicate that these residues interact differently with these closely related drugs.

  • identification of a Phenylalkylamine binding region within the alpha 1 subunit of skeletal muscle ca2 channels
    Proceedings of the National Academy of Sciences of the United States of America, 1990
    Co-Authors: J. Striessnig, Hartmut Glossmann, William A Catterall
    Abstract:

    Abstract The alpha 1 subunit of the skeletal muscle Ca2+ channel has been specifically photoaffinity labeled with the Phenylalkylamine-receptor-selective verapamil derivative (-)-5-(3-azidophenethyl[N-methyl-3H]methylamino)-2-(3,4,5- trimethoxyphenyl)-2-isopropylvaleronitrile ([N-methyl-3H]LU49888). Proteolytic fragments generated by various endoproteases were probed by immunoprecipitation with several sequence-specific antibodies to determine the site of labeling within the primary structure of alpha 1. These results restrict the site of photolabeling by [N-methyl-3H]LU49888 to the region between Glu-1349 and Trp-1391. This segment of alpha 1 contains transmembrane helix S6 of domain IV and the beginning of the long intracellular C-terminal tail. Because of the Phenylalkylamine receptor site is only accessible from the intracellular side of the Ca2+ channel, we propose that the intracellular end of helix IVS6 and the adjacent intracellular amino acid residues play an essential role in formation of the Phenylalkylamine receptor site. The action of the Phenylalkylamines as open-channel blockers suggests that this region may also contribute to formation of the intracellular opening of the transmembrane pore of the Ca2+ channel.

J. Striessnig - One of the best experts on this subject based on the ideXlab platform.

  • opposite effects of a single iiis5 mutation on Phenylalkylamine and dihydropyridine interaction with l type ca2 channels
    Journal of Biological Chemistry, 2004
    Co-Authors: Irene G Huber, Edwin Wapplkornherr, Martina J Sinneggerbrauns, Jeancharles Hoda, Doris Walterbastl, J. Striessnig
    Abstract:

    Abstract Replacement of L-type Ca2+ channel α1 subunit residue Thr-1066 in segment IIIS5 by a tyrosine residue conserved in the corresponding positions of non-L-type Ca2+ channels eliminates high dihydropyridine sensitivity through a steric mechanism. To determine the effects of this mutation on Phenylalkylamine interaction, we exploited the availability of Cav1.2DHP–/– mice containing the T1066Y mutation. In contrast to dihydropyridines, increased protein-dependent binding of the Phenylalkylamine (–)-[3H]devapamil occurred to Cav1.2DHP–/– mouse brain microsomes. This effect could be attributed to an at least 2-fold increase in affinity as determined by saturation analysis and binding inhibition experiments. The latter also revealed a higher affinity for (–)-verapamil but not for (–)-gallopamil. The mutation caused a pronounced slowing of (–)-[3H]devapamil dissociation, indicating a stabilization of the drug-channel complex. The increased affinity of mutant channels was also evident in functional studies after heterologous expression of wild type and T1066Y channels in Xenopus laevis oocytes. 100 μm (–)-verapamil inhibited a significantly larger fraction of Ba2+ inward current through mutant than through WT channels. Our results provide evidence that Phenylalkylamines also interact with the IIIS5 helix and that the geometry of the IIIS5 helix affects the access and/or binding of different chemical classes of Ca2+ channel blockers to their overlapping binding domains. Mutation of Thr-1066 to a non-L-type tyrosine residue can be exploited to differentially affect Phenylalkylamine and dihydropyridine binding to L-type Ca2+ channels.

  • identification of a 27 kda high affinity Phenylalkylamine binding polypeptide as the sigma 1 binding site by photoaffinity labeling and ligand directed antibodies
    Molecular Pharmacology, 1993
    Co-Authors: Fabian F Moebius, J. Striessnig, Gregory G Burrows, Markus Hanner, Eduard Schmid, Hartmut Glossmann
    Abstract:

    The verapamil-like arylazide (-)-[3H]azidopamil specifically photoaffinity labeled two low molecular mass polypeptides, with apparent molecular masses of 22 and 27 kDa, in the endoplasmic reticulum of guinea pig liver, kidney, adrenal gland, and lung. It was recently shown that the 22-kDa polypeptide binds the anti-ischemic Phenylalkylamine (-)-[3H]emopamil and other anti-ischemic drugs with high affinity. We now provide evidence that the photolabeling of the 27-kDa polypeptide is blocked by nanomolar concentrations of sigma ligands [order of potency, haloperidol > pentazocine > 1,3-ditolylguanidine > dextromethorphan > (+)-SKF10,047]. The apparent affinities of these and other drugs closely corresponded to those for 1,3-[3H]ditolylguanidine-labeled sigma binding sites. Based on its high affinity for the (+)-enantiomer [but not the (-)-enantiomer] of SKF10,047 (Ki = 51 nM), pentazocine (Ki = 3 nM), and dextromethorphan (Ki = 30 nM), the (-)-[3H]azidopamil-labeled site on the 27-kDa polypeptide was classified as being of the sigma 1 subtype. Using antiPhenylalkylamine antibodies, we developed a novel immunological detection method that allows the rapid and sensitive staining of the photolabeled 27-kDa polypeptide after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We conclude that the Phenylalkylamines emopamil and azidopamil represent a novel class of sigma ligands, highly suitable for the further structural characterization of polypeptides carrying sigma 1 binding sites.

  • Very high affinity interaction of DPI 201-106 and BDF 8784 enantiomers with the Phenylalkylamine-sensitive Ca2+-channel in Drosophila head membranes
    British Journal of Pharmacology, 1991
    Co-Authors: Hartmut Glossmann, R. Staudinger, J. Striessnig, R. Greenberg, Christian Zech, L Hall, B. I. Armah
    Abstract:

    1. Piperazinylindoles (DPI 201-106, BDF 8784), drugs known to act on voltage-dependent Na(+)-channels, bind with very high affinity to a Ca2(+)-channel-associated Phenylalkylamine receptor in Drosophila melanogaster head membranes. These compounds and (+)-tetrandrine, a naturally occurring Ca2(+)-antagonist, were the most selective inhibitors for Phenylalkylamine-labelled Drosophila Ca2(+)-channels compared to mammalian L-type Ca2(+)-channels. 2. Replacement of the cyano group by a methyl group in (+)-DPI 201-106 ((+)-BDF 8784) increases the IC50 value for inhibition of Phenylalkylamine labelling of Drosophila Ca2(+)-channels from 0.29 to 2.1 nM but decreases the IC50 value for inhibition of Phenylalkylamine labelling of mammalian skeletal muscle Ca2(+)-channels from 3480 to 49 nM. 3. DPI 201-106 enantiomers completely block (at 0.1 microM) Phenylalkylamine photolabelling of a 136 K polypeptide in Drosophila head membranes whereas 10 microM aconitine or lidocaine are without effect. 4. Assessment of the Ca2(+)-antagonist effects of the substituted DPI 201-106 enantiomers in K(+)-depolarized taenia strips from guinea-pig caecum yielded pA2 values of 6.33 +/- 0.07 for (-)-BDF 8784 and 6.99 +/- 0.17 for (+)-BDF 8784, respectively. 5. Piperazinylindoles, previously believed to act nonspecifically on voltage-dependent mammalian L-type Ca2(+)-channels, therefore have stereoselectivity for a novel binding site and chemical selectivity unrelated to local anaesthetic activity. 6. It is proposed that a very high affinity piperazinylindole-selective site is coupled to the Phenylalkylamine receptor of Drosophila Ca2(+)-channels. These sites are still present on mammalian L-type Ca2(+)-channels but have lower affinity and/or are less tightly coupled to Phenylalkylamine receptors on the alpha 1-subunit.

  • identification of a Phenylalkylamine binding region within the alpha 1 subunit of skeletal muscle ca2 channels
    Proceedings of the National Academy of Sciences of the United States of America, 1990
    Co-Authors: J. Striessnig, Hartmut Glossmann, William A Catterall
    Abstract:

    Abstract The alpha 1 subunit of the skeletal muscle Ca2+ channel has been specifically photoaffinity labeled with the Phenylalkylamine-receptor-selective verapamil derivative (-)-5-(3-azidophenethyl[N-methyl-3H]methylamino)-2-(3,4,5- trimethoxyphenyl)-2-isopropylvaleronitrile ([N-methyl-3H]LU49888). Proteolytic fragments generated by various endoproteases were probed by immunoprecipitation with several sequence-specific antibodies to determine the site of labeling within the primary structure of alpha 1. These results restrict the site of photolabeling by [N-methyl-3H]LU49888 to the region between Glu-1349 and Trp-1391. This segment of alpha 1 contains transmembrane helix S6 of domain IV and the beginning of the long intracellular C-terminal tail. Because of the Phenylalkylamine receptor site is only accessible from the intracellular side of the Ca2+ channel, we propose that the intracellular end of helix IVS6 and the adjacent intracellular amino acid residues play an essential role in formation of the Phenylalkylamine receptor site. The action of the Phenylalkylamines as open-channel blockers suggests that this region may also contribute to formation of the intracellular opening of the transmembrane pore of the Ca2+ channel.

Moon Kyo In - One of the best experts on this subject based on the ideXlab platform.

  • rapid and simple gc ms method for determination of psychotropic Phenylalkylamine derivatives in nails using micro pulverized extraction
    Journal of Forensic Sciences, 2012
    Co-Authors: Jae Chul Cheong, Moon Kyo In
    Abstract:

    :  A rapid and simple gas chromatography–mass spectrometry (GC–MS) method was developed and validated for the simultaneous detection and quantification of five psychotropic Phenylalkylamines (amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, and norketamine) in toenails. After external decontamination, nail clippings were mechanically pulverized with a bead mill and then incubated in methanol under ultrasonication at 50°C for 1 h. The resulting solutions were evaporated to dryness, derivatized, and analyzed by GC–MS. The intra- and inter-day precisions were within 10.7% and 13.9%, respectively. The intra- and inter-day accuracies were −4.2% to 5.0% and −2.4% to 8.4%, respectively. Limits of detection and quantification for each analyte were lower than 0.024 and 0.08 ng/mg, respectively. The recoveries were in the range of 80.6–87.5%. The results indicated that the proposed method is a simple, rapid, accurate, and precise for quantification of five Phenylalkylamines in nails. The method was successfully applied to the simultaneous detection and quantification of Phenylalkylamines in nail samples of possible drug abusers.

  • Rapid and Simple GC–MS Method for Determination of Psychotropic Phenylalkylamine Derivatives in Nails Using Micro‐Pulverized Extraction*
    Journal of Forensic Sciences, 2011
    Co-Authors: Jae Chul Cheong, Moon Kyo In
    Abstract:

    :  A rapid and simple gas chromatography–mass spectrometry (GC–MS) method was developed and validated for the simultaneous detection and quantification of five psychotropic Phenylalkylamines (amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, and norketamine) in toenails. After external decontamination, nail clippings were mechanically pulverized with a bead mill and then incubated in methanol under ultrasonication at 50°C for 1 h. The resulting solutions were evaporated to dryness, derivatized, and analyzed by GC–MS. The intra- and inter-day precisions were within 10.7% and 13.9%, respectively. The intra- and inter-day accuracies were −4.2% to 5.0% and −2.4% to 8.4%, respectively. Limits of detection and quantification for each analyte were lower than 0.024 and 0.08 ng/mg, respectively. The recoveries were in the range of 80.6–87.5%. The results indicated that the proposed method is a simple, rapid, accurate, and precise for quantification of five Phenylalkylamines in nails. The method was successfully applied to the simultaneous detection and quantification of Phenylalkylamines in nail samples of possible drug abusers.

  • rapid and simple determination of psychotropic Phenylalkylamine derivatives in human hair by gas chromatography mass spectrometry using micro pulverized extraction
    Forensic Science International, 2010
    Co-Authors: Soon Ho Shin, Moon Kyo In
    Abstract:

    Abstract A gas chromatography–mass spectrometric (GC–MS) method was developed and validated for the determination of five psychotropic Phenylalkylamine derivatives (amphetamine, AP; methamphetamine, MA; 3,4-methylenedioxyamphetamine, MDA; 3,4-methylenedioxymethamphetamine, MDMA; norketamine, NKT) in human hair. Hair samples (10 mg) were washed with distilled water and acetone, mechanically pulverized for 1.5 min with a bead mill, and then incubated in 1 mL of methanol under ultrasonication at 50 °C for 1 h. The resulting solutions were evaporated to dryness, derivatized using heptafluorobutyric anhydride (HFBA) at 50 °C for 30 min, and analyzed by GC–MS. The linear ranges were 0.1–20.0 ng/mg for AP and MA and 0.05–20.0 ng/mg for MDA, MDMA, and NKT, with the coefficients of determination ( r 2  > 0.9982). The intra-day and inter-day precisions were within 11.5% and 12.8%, respectively. The intra-day and inter-day accuracies were −4.1% to 5.8% and −6.6% to 4.2%, respectively. The limits of detections (LODs) for each compound were lower than 0.028 ng/mg. The recoveries were in the range of 78.9–101.2%. Based on these results, the method proved to be effective for the rapid and simple determination of Phenylalkylamine derivatives in hair specimens.

  • Rapid and simple determination of psychotropic Phenylalkylamine derivatives in human hair by gas chromatography-mass spectrometry using micro-pulverized extraction.
    Forensic science international, 2010
    Co-Authors: Soon Ho Shin, Moon Kyo In
    Abstract:

    A gas chromatography-mass spectrometric (GC-MS) method was developed and validated for the determination of five psychotropic Phenylalkylamine derivatives (amphetamine, AP; methamphetamine, MA; 3,4-methylenedioxyamphetamine, MDA; 3,4-methylenedioxymethamphetamine, MDMA; norketamine, NKT) in human hair. Hair samples (10mg) were washed with distilled water and acetone, mechanically pulverized for 1.5 min with a bead mill, and then incubated in 1 mL of methanol under ultrasonication at 50 degrees C for 1h. The resulting solutions were evaporated to dryness, derivatized using heptafluorobutyric anhydride (HFBA) at 50 degrees C for 30 min, and analyzed by GC-MS. The linear ranges were 0.1-20.0 ng/mg for AP and MA and 0.05-20.0 ng/mg for MDA, MDMA, and NKT, with the coefficients of determination (r(2)>0.9982). The intra-day and inter-day precisions were within 11.5% and 12.8%, respectively. The intra-day and inter-day accuracies were -4.1% to 5.8% and -6.6% to 4.2%, respectively. The limits of detections (LODs) for each compound were lower than 0.028 ng/mg. The recoveries were in the range of 78.9-101.2%. Based on these results, the method proved to be effective for the rapid and simple determination of Phenylalkylamine derivatives in hair specimens.

  • simultaneous determination of psychotropic Phenylalkylamine derivatives in human hair by gas chromatography mass spectrometry
    Rapid Communications in Mass Spectrometry, 2007
    Co-Authors: Kyu Sung Jung, Moon Kyo In
    Abstract:

    A gas chromatography/mass spectrometric (GC/MS) method was developed and validated for the determination of thirteen psychotropic Phenylalkylamine derivatives (amphetamine; AP, phentermine; PT, methamphamine; MA, cathinone; Khat, methcathinone; MCAT, fenfluramine; FFA, desmethylselegiline; DSEL, 3,4-methylenedioxyamphetamine; MDA, 3,4-methylenedioxymethamphetamine; MDMA, 3,4-methylenedioxyethylamphetamine; MDEA, norketamine; NKT, mescaline; MES, 4-bromo-2,5-dimethoxyphenethylamine; 2CB) in human hair. Hair samples (20 mg) were washed with distilled water and acetone, cut into small fragments ( 0.9985). The intra-day, inter-day, and inter-person precisions were within 12.7%, 14.8%, and 16.8%, respectively. The intra-day, inter-day, and inter-person accuracies were between -10.7 and 13.4%, -12.7 and 11.6%, and -15.3 and 11.9%, respectively. The limits of quantifications (LOQs) for each compound were lower than 0.08 ng/mg. The recoveries were in the range of 76.7-95.6%. The method proved to be suitable for the simultaneous qualification and quantification of Phenylalkylamine derivatives in hair specimens.

Gregory H. Hockerman - One of the best experts on this subject based on the ideXlab platform.

  • molecular determinants of high affinity Phenylalkylamine block of l type calcium channels in transmembrane segment iiis6 and the pore region of the α1subunit
    Journal of Biological Chemistry, 1997
    Co-Authors: Gregory H. Hockerman, Barry D. Johnson, Todd Scheuer, Michael R Abbott, William A Catterall
    Abstract:

    Abstract Recent studies of the Phenylalkylamine binding site in the α1C subunit of l-type Ca2+ channels have revealed three amino acid residues in transmembrane segment IVS6 that are critical for high affinity block and are unique to l-type channels. We have extended this analysis of the Phenylalkylamine binding site to amino acid residues in transmembrane segment IIIS6 and the pore region. Twenty-two consecutive amino acid residues in segment IIIS6 were mutated to alanine and the conserved Glu residues in the pore region of each homologous domain were mutated to Gln. Mutant channels were expressed in tsA-201 cells along with the β1b and α2δ auxiliary subunits. Assay for block of Ba2+ current by (−)-D888 at −60 mV revealed that mutation of five amino acid residues in segment IIIS6 and the pore region that are conserved between l-type and non-l-type channels (Tyr1152, Phe1164, Val1165, Glu1118, and Glu1419) and one l-type-specific amino acid (Ile1153) decreased affinity for (−)-D888 from 10–20-fold. Combination of the four mutations in segment IIIS6 increased the IC50 for block by (−)-D888 to approximately 9 μm, similar to the affinity of non-l-type Ca2+ channels for this drug. These results indicate that there are important determinants of Phenylalkylamine binding in both the S6 segments and the pore regions of domains III and IV, some of which are conserved across the different classes of voltage-gated Ca2+ channels. A model of the Phenylalkylamine receptor site at the interface between domains III and IV of the α1 subunit is presented.

  • Distinct effects of mutations in transmembrane segment IVS6 on block of L-type calcium channels by structurally similar Phenylalkylamines.
    Molecular Pharmacology, 1996
    Co-Authors: Barry D. Johnson, Gregory H. Hockerman, Todd Scheuer, William A Catterall
    Abstract:

    The Phenylalkylamines (-)-D888, verapamil, and D600, cause voltage- and use-dependent block of L-type Ca2+ channels and differ from each other only in the number of methoxy groups on each of their two terminal phenyl rings. To study the effects of mutations in the Phenylalkylamine receptor site on block by these drugs, wild-type and mutant Ca2+ channels were transiently expressed in the tsA-201 clone of human embryonic kidney 293 cells. The combined mutations Y1463A, A1467S, and I1470A (mutant YAI) in transmembrane segment S6 of domain IV of the alpha 1c subunit disrupted block by all three Phenylalkylamines. Surprisingly, although this mutation reduced both resting block at -60 mV and depolarized block at +10 mV by (-)-D888, resting and depolarized block by verapamil and D600 were relatively unaffected. In contrast, for all three drugs, use-dependent block during repetitive stimulations was sharply reduced, and the rate of recovery from depolarized block was accelerated for YAI channels. Thus, the effects of the YAI mutation on apparent affinity were specific to (-)-D888, whereas effects on the kinetics of block were observed for all three drugs. Additional experiments with substitution of phenylalanine for Y1463 suggested that (-)-D888 affinity is specifically sensitive to removal of the hydroxyl group of Y1463, whereas effects on the kinetics of block by all three Phenylalkylamines require larger molecular changes, perhaps related to residue size and hydrophobicity. Analysis of the data using a state-dependent model of drug block suggests that these kinetic differences are caused by both changes in drug access to the receptor site and affinity for binding to the inactivated state of the channel. The different effects of the YAI mutations on the actions of (-)-D888, verapamil, and D600 indicate that these residues interact differently with these closely related drugs.