µ-Theraphotoxin-Pn3a inhibition of CaV3.3 channels reveals a novel isoform-selective drug binding site
Abstract
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. We have identified μ-theraphotoxin 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, site-directed mutagenesis, and full domain II swaps between Cav3 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 CaV3.3 channels.
Data availability
All data generated or analysed during this study are included in the manuscript and supporting file
Article and author information
Author details
Funding
Rebecca L. Cooper Medical Research Foundation (PG2019396)
- Jeffrey R McArthur
National Health and Medical Research Council (APP1072113)
- David J Adams
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Henry M Colecraft, Columbia University, United States
Version history
- Received: September 20, 2021
- Preprint posted: October 4, 2021 (view preprint)
- Accepted: July 19, 2022
- Accepted Manuscript published: July 20, 2022 (version 1)
- Version of Record published: August 1, 2022 (version 2)
Copyright
© 2022, McArthur et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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