Dutch Boltz - Molecular Devices

Abstract
Ion channels are an attractive class of drug targets because of their involvement in a diverse range of
cardiovascular, inflammatory, and neurological disorders. Their pharmacological complexity proves
advantageous for developing highly selective drugs that affect only specific functional states of the channel.
Advancements in fluorescent dyes and optical detection technologies have provided drug discovery programs
with functional screening assays capable of detecting ion channel modulation in an HTS format. We have
developed a membrane potential kinetics screen for a FLIPR-384 instrument that utilizes a FRET-based
coumarin-DiSBAC 2
dye system to screen inhibitors of ion channel X expressed in HEK293 cells. We have
modified a FLIPR-384 instrument with a 409nm krypton laser, introducing the beam co-linear to the 488nm
argon laser path by additional beam steering optics in order to overcome limitations which previously
restricted the use of such FRET based dyes on FLIPR . A number of channel X specific compounds were
screened on the modified FLIPR-384 and compared to results generated on the standard kinetic plate reader.
There was good correlation between both systems for all tested compounds. The modified FLIPR had a
slightly increased sensitivity and a 12-24 factor increase in throughput. Running this assay in ratiometric
mode on FLIPR, reading all 384 wells simultaneously, eliminates the run-down of signal and variability
observed on the other plate reader. These findings demonstrate the feasibility of using a modified FLIPR-384
as an HTS platform utilizing FRET based voltage sensors to identify novel ion channel modulators.
Additionally, the same configuration with an argon-krypton laser substitution for the argon primary laser
would permit the use of virtually any dye on FLIPR with the possibility of simultaneously following two
distinct physiological sensors .
Introduction
Historically, ion channel HTS assays have taken the form of membrane potential or calcium flux population
kinetics. More recent approaches have shifted toward the use of membrane potential sensitive fluorophores
that undergo redistribution within the plasma membrane in response to voltage changes. One such approach
makes use of DiSBAC 4
, or a similar single excitation, single emission dye to measure time courses greater than
10 seconds. A more rapid technique was later developed that utilizes a Coumarin-DiSBAC 2
FRET dye pair to
deal with time courses less than 10 seconds. It has the distinct advantage of functioning as a ratiometric dye
with increased temporal resolution, reproducibility and throughput; advancing measurements beyond the
detection of steady-state changes in membrane potential and avoiding the need for pharmacological
6/14/2004 Cardiovascular Diseases 23
modification of rapidly inactivated or desensitized ion channels.