α-Conotoxins selectively bind to nicotinic acetylcholine receptors (nAChRs), which are therapeutic targets due to their important role in signaling transmission in excitable cells. A previous experimental study has demonstrated that carboxylation of the C-terminal of α-conotoxin LsIA reduces its potency to inhibit human α7 nAChR relative to naturally amidated LsIA. However, little is known about the contribution of conformational changes in the receptor and interactions, induced by C-terminal amidation/carboxylation of conotoxins, to selective binding to nAChRs, since most conotoxins and some disulfide-rich peptides from other conotoxin subfamilies possess a naturally amidated C-terminal. In this study, we employ homology modeling and molecular dynamics (MD) simulations to propose the determinants for differential interactions between amidated and carboxylated LsIAs with α7 nAChR. Our findings indicate an overall increased number of contacts favored by binding of amidated LsIA versus its carboxylated counterpart. Toxin-receptor pairwise interactions, which may play a role in enhancing the potency of the former, include ARG10-TRP77, LEU141 and CYS17-GLN79 via persistent hydrogen bonds and cation-π interactions, which are weakened in the carboxylated form due to a strong intramolecular salt-bridge formed by ARG10 and carboxylated C-terminus. The binding of amidated LsIA also induces enhanced movements in loop C and the juxtamembrane Cys-loop that are closely associated with receptor function. Additionally, the impacts of binding of LsIA on the overall structure and inter-subunit contacts were examined using inter-residue network analysis, suggesting a clockwise tilting of the α7 C and F loops upon binding to carboxylated LsIA, which is absent for amidated LsIA binding. The predicted molecular mechanism of LsIA binding to the α7 receptor may provide new insights into the important role of the C-terminal in the binding potency of conotoxins at neuronal nAChRs for pharmacological purposes.
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.
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