μ-Theraphotoxin-Pn3a inhibition of Ca _V 3.3 channels reveals a novel isoform-selective drug binding site

Low voltage-activated calcium currents are mediated by T-type calcium channels Ca _V 3.1, Ca _V 3.2, and Ca _V 3.3, which modulate a variety of physiological processes including sleep, cardiac pace-making, pain, and epilepsy. Ca _V 3 isoforms’ biophysical properties, overlapping expression and lack of subtype-selective pharmacology hinder the determination of their specific physiological roles in health and disease. Notably, Ca _V 3.3’s contribution to normal and pathophysiological function has remained largely unexplored. We have identified Pn3a as the first subtype-selective spider venom peptide inhibitor of Ca _V 3.3, with >100-fold lower potency against the other T-type isoforms. Pn3a modifies Ca _V 3.3 gating through a depolarizing shift in the voltage dependence of activation thus decreasing Ca _V 3.3-mediated currents in the normal range of activation potentials. Paddle chimeras of K _V 1.7 channels bearing voltage sensor sequences from all four Ca _V 3.3 domains revealed preferential binding of Pn3a to the S3-S4 region of domain II (Ca _V 3.3 ^DII ). 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 Ca _V 3.3 channels.