OBJECTIVE: The aim of this study was to compare a diverse set of peptide and small-molecule calcium channel blockers for inactivated-state block of native and recombinant N-type calcium channels using fluorescence-based and automated patch-clamp electrophysiology assays. METHODS: The pharmacology of calcium channel blockers was determined at N-type channels in IMR-32 cells and in HEK cells overexpressing the inward rectifying K(+) channel Kir2.1. N-type channels were opened by increasing extracellular KCl. In the Kir2.1/N-type cell line the membrane potential could be modulated by adjusting the extracellular KCl, allowing determination of resting and inactivated-state block of N-type calcium channels. The potency and degree of state-dependent inhibition of these blockers were also determined by automated patch-clamp electrophysiology. RESULTS: N-type-mediated calcium influx in IMR-32 cells was determined for a panel of blockers with IC(50) values of 0.001-7 μM and this positively correlated with inactivated-state block of recombinant channels measured using electrophysiology. The potency of several compounds was markedly weaker in the state-dependent fluorescence-based assay compared to the electrophysiology assay, although the degree of state-dependent blockade was comparable. CONCLUSIONS: The present data demonstrate that fluorescence-based assays are suitable for assessing the ability of blockers to selectively interact with the inactivated state of the N-type channel.
OBJECTIVE: The aim of this study was to compare a diverse set of peptide and small-molecule calcium channel blockers for inactivated-state block of native and recombinant N-type calcium channels using fluorescence-based and automated patch-clamp electrophysiology assays. METHODS: The pharmacology of calcium channel blockers was determined at N-type channels in IMR-32 cells and in HEK cells overexpressing the inward rectifying K(+) channel Kir2.1. N-type channels were opened by increasing extracellular KCl. In the Kir2.1/N-type cell line the membrane potential could be modulated by adjusting the extracellular KCl, allowing determination of resting and inactivated-state block of N-type calcium channels. The potency and degree of state-dependent inhibition of these blockers were also determined by automated patch-clamp electrophysiology. RESULTS: N-type-mediated calcium influx in IMR-32 cells was determined for a panel of blockers with IC(50) values of 0.001-7 μM and this positively correlated with inactivated-state block of recombinant channels measured using electrophysiology. The potency of several compounds was markedly weaker in the state-dependent fluorescence-based assay compared to the electrophysiology assay, although the degree of state-dependent blockade was comparable. CONCLUSIONS: The present data demonstrate that fluorescence-based assays are suitable for assessing the ability of blockers to selectively interact with the inactivated state of the N-type channel.
Authors: H Saegusa; T Kurihara; S Zong; A Kazuno ; Y Matsuda; T Nonaka; W Han; H Toriyama; T Tanabe Journal: EMBO J Date: 2001-05-15 Impact factor: 11.598
Authors: Francesco Belardetti; Elizabeth Tringham; Cyrus Eduljee; Xinpo Jiang; Haiheng Dong; Adam Hendricson; Yoko Shimizu; Diana L Janke; David Parker; Janette Mezeyova; Afsheen Khawaja; Hassan Pajouhesh; Robert A Fraser; Stephen P Arneric; Terrance P Snutch Journal: Assay Drug Dev Technol Date: 2009-06 Impact factor: 1.738
Authors: Hassan Pajouhesh; Zhong-Ping Feng; Yanbing Ding; Lingyun Zhang; Hossein Pajouhesh; Jerrie-Lynn Morrison; Francesco Belardetti; Elizabeth Tringham; Eric Simonson; Todd W Vanderah; Frank Porreca; Gerald W Zamponi; Lester A Mitscher; Terrance P Snutch Journal: Bioorg Med Chem Lett Date: 2010-01-07 Impact factor: 2.823