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. We have identified μ-theraphotoxin 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, site-directed mutagenesis, and full domain II swaps between Ca(V)3 channels highlighting it as a subtype-specific pharmacophore. 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.