{micro}-Theraphotoxin-Pn3a inhibition of Cav3.3 channels reveals a novel isoform-selective drug binding site

Low voltage-activated calcium currents are mediated by T-type calcium channels CaV3.1, CaV3.2, and CaV3.3, which modulate a variety of physiological processes including sleep, cardiac pace-making, pain, and epilepsy. CaV3 isoforms biophysical properties, overlapping expression and lack of subtype-selective pharmacology hinder the determination of their specific physiological roles in health and disease. Notably, CaV3.3s contribution to normal and pathophysiological function has remained largely unexplored. We have identified Pn3a as the first subtype-selective spider venom peptide inhibitor of CaV3.3, with >100-fold lower potency against the other T-type isoforms. Pn3a modifies CaV3.3 gating through a depolarizing shift in the voltage dependence of activation thus decreasing CaV3.3-mediated currents in the normal range of activation potentials. Paddle chimeras of KV1.7 channels bearing voltage sensor sequences from all four CaV3.3 domains revealed preferential binding of Pn3a to the S3-S4 region of domain II (CaV3.3DII). This novel T-type channel pharmacological site was explored through computational docking simulations of Pn3a into all T-type channel isoforms highlighting it as subtype-specific pharmacophore with therapeutic potential. This research expands our understanding of T-type calcium channel pharmacology and supports the suitability of Pn3a as a molecular tool in the study of the physiological roles of CaV3.3 channels.