State-dependent inhibition of L-type calcium channels: cell-based assay in high-throughput format

Abstract

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 Ca 2+ influx through the L-type Ca 2+ channel complex (α1C, α2δ, and β2a subunits) in response to potassium-mediated depolarization; EC 50=11 mM [K +] o. The Ca 2+ influx was inhibited by the L-type Ca 2+ 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 Ca 2+ channel is altered under these different conditions. Under the depolarized condition, nimodipine was a more potent antagonist, inhibiting Ca 2+ 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.