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 muM 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.
        
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Vortherms TA, Swensen AM, Niforatos W, Limberis JT, Neelands TR, Janis RS, Thimmapaya R, Donnelly-Roberts D, Namovic MT, Zhang D, Brent Putman C, Martin RL, Surowy CS, Jarvis MF, Scott VE (2011) Comparative analysis of inactivated-state block of N-type (Ca(v)2.2) calcium channels Inflamm Res60: 683-93
Vortherms TA, Swensen AM, Niforatos W, Limberis JT, Neelands TR, Janis RS, Thimmapaya R, Donnelly-Roberts D, Namovic MT, Zhang D, Brent Putman C, Martin RL, Surowy CS, Jarvis MF, Scott VE (2011) Inflamm Res60: 683-93