State-dependent inhibition of L-type calcium channels: cell-based assay in high-throughput format.
The current studies describe a new, robust cell-based functional assay useful to characterize L-type voltage-dependent calcium channels and their antagonists. The basis of this assay is measurement in plate format of Ca2+ influx through the L-type Ca2+ channel complex (alpha1C, alpha2delta, and beta2a subunits) in response to potassium-mediated depolarization; EC(50)=11 mM [K+](o). The Ca2+ influx was inhibited by the L-type Ca2+ channel antagonist, nimodipine; IC(50)=59 nM. These cells were also transfected with the Kir2.3 inward rectifier K(+) channel, which allows for changing the cell membrane potential by modulation of extracellular [K](o); -65 mV in physiological [K](o) and -28 mV in 30 mM [K](o) containing buffer. The conformational state of the voltage-sensitive Ca2+ channel is altered under these different conditions. Under the depolarized condition, nimodipine was a more potent antagonist, inhibiting Ca2+ influx with an IC(50) value of 3 nM. The results demonstrate that the interaction of nimodipine and other antagonists with the channel is modulated by changes in membrane potential and thus the state of the channel. Overall, this novel assay can be used to identify state-dependent calcium channel antagonists and should be useful for evaluating state-dependent inhibitory potency of a large number of samples.
Xia
Menghang
M
Department of Molecular Pharmacology, Merck Research Laboratories, West Point, PA 19486, USA. menghang_xia@merck.com
Imredy
John P
JP
Koblan
Ken S
KS
Bennett
Paul
P
Connolly
Thomas M
TM
D001681:Biological Assay / Q000379:methods*; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D015220:Calcium Channels / Q000378:metabolism; D020746:Calcium Channels, L-Type / Q000378:metabolism*; D002460:Cell Line; D004594:Electrophysiology; D005470:Fluorometry; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects; D009553:Nimodipine / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism / Q000494:pharmacology; D024661:Potassium Channels, Inwardly Rectifying; D011506:Proteins / Q000378:metabolism
C480968:CACNA2D2 protein, human; D002121:Calcium Channel Blockers; D015220:Calcium Channels; D020746:Calcium Channels, L-Type; C099489:KCNJ4 protein, human; C419385:L-type calcium channel alpha(1C); D024661:Potassium Channels, Inwardly Rectifying; D011506:Proteins; D009553:Nimodipine; D011188:Potassium