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Inflammation Research
Published in: Inflammation Research 7/2011

01-07-2011 | Original Research Paper

Comparative analysis of inactivated-state block of N-type (Cav2.2) calcium channels

Authors: Timothy A. Vortherms, Andrew M. Swensen, Wende Niforatos, James T. Limberis, Torben R. Neelands, Richard S. Janis, Rama Thimmapaya, Diana L. Donnelly-Roberts, Marian T. Namovic, Di Zhang, C. Brent Putman, Ruth L. Martin, Carol S. Surowy, Michael F. Jarvis, Victoria E. Scott

Published in: Inflammation Research | Issue 7/2011

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Abstract

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 IC50 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.
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Metadata
Title
Comparative analysis of inactivated-state block of N-type (Cav2.2) calcium channels
Authors
Timothy A. Vortherms
Andrew M. Swensen
Wende Niforatos
James T. Limberis
Torben R. Neelands
Richard S. Janis
Rama Thimmapaya
Diana L. Donnelly-Roberts
Marian T. Namovic
Di Zhang
C. Brent Putman
Ruth L. Martin
Carol S. Surowy
Michael F. Jarvis
Victoria E. Scott
Publication date
01-07-2011
Publisher
SP Birkhäuser Verlag Basel
Published in
Inflammation Research / Issue 7/2011
Print ISSN: 1023-3830
Electronic ISSN: 1420-908X
DOI
https://doi.org/10.1007/s00011-011-0322-9

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