Establishment of a secondary screening assay for P/Q-type calcium channel blockers.
2013
Development of calcium channel blockers is attractive, but has in the past been hampered by lack of high
throughput electrophysiological technology. This limitation has been overcome by the implementation of automated patch
clamp systems that allow identification of state-dependent compounds, which preferentially target pathologically
overactive channels.
We recently presented a fluorescence-based high-throughput screen for P/Q-type calcium channels followed by automated
electrophysiology. Here, we provide a detailed description of the development of the secondary screen, and show the full
analysis of the inactivation kinetics of the recombinant P/Q channel that served as a basis for the automated patch clamp
protocol. Increasing the length of pre-depolarization shifted the inactivation to more hyperpolarized potentials. No steadystate
inactivation was reached up to pre-depolarization durations of 3 min, while stability of the recordings progressively
declined. As a compromise, a 3s pre-depolarization protocol was proposed for functional screening. In order to validate
the electrophysiological screening, we compared kinetics and pharmacology of recombinant P/Q-type channels between
automated and manual patch clamp measurements. Channel activation was similar under both conditions. By contrast,
inactivation occurred at more hyperpolarized potentials in the automated system. Therefore, P/Q-type calcium channel
inactivation is sensitive to the applied technological platform and needs to be adjusted when performing automated patch
clamp recordings.
Our results indicate that a thorough analysis of the inactivation kinetics is mandatory, when establishing an
electrophysiological screening protocol for calcium channel blockers. As some data obtained by automated recordings
may not be identical to manual patch clamp analysis, we recommend a proper initial validation of the screening assay and
– if necessary – a posthoc adjustment of automated patch clamp values. The protocol presented here supports hit-to-lead
and lead optimization efforts during the development of novel P/Q-type calcium channel blockers, and may be valuable
for the generation of assays in other ion channel programs.
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