An Automated Electrophysiological Assay for Differentiating CaV2.2 Inhibitors Based on State Dependence and Kinetics

Abstract CaV2.2 (N-type) calcium channels are key regulators of neurotransmission. Evidence from knockout animals and localization studies suggest that CaV2.2 channels play a critical role in nociceptive transmission. Additionally, ziconotide, a selective peptide inhibitor of CaV2.2 channels, is clinically used to treat refractory pain. However, the use of ziconotide is limited by its low therapeutic index, which is believed, at least in part, to be a consequence of ziconotide inhibiting CaV2.2 channels regardless of the channel state. Subsequent efforts have focused on the discovery of state-dependent inhibitors that preferentially bind to the inactivated state of CaV2.2 channels in order to achieve an improved safety profile relative to ziconotide. Much less attention has been paid to understanding the binding kinetics of these state-dependent inhibitors. Here, we describe a novel electrophysiology-based assay on an automated patch platform designed to differentiate CaV2.2 inhibitors based on their comb...