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.

Pmu1a, a novel spider toxin with dual inhibitory activity at pain targets hNaV1.7 and hCaV3 voltage‐gated channels

“…The majority of Ca V active spider peptides have shown activity across Ca V 1, Ca V 2.1, Ca V 2.2 and Ca V 2.3 [42–56], with a recent report of the tarantula toxin ω‐Avsp1a inhibiting T‐type calcium channels Ca V 3.1 and Ca V 3.3, albeit with low affinity [57]. Recently, low potency inhibition of calcium channels Ca V 3.3 by the spider toxin Pn3a, a low nanomolar inhibitor of Na V 1.7 sodium channels, was reported [13]. However, the authors did not observe Ca V 3.2 inhibition up to 10 μ m [13].…”

“…Recently, low potency inhibition of calcium channels Ca V 3.3 by the spider toxin Pn3a, a low nanomolar inhibitor of Na V 1.7 sodium channels, was reported [13]. However, the authors did not observe Ca V 3.2 inhibition up to 10 μ m [13]. Tap1a isolated from Theraphosa apophysis venom is thus far the spider toxin displaying the strongest activity on Ca V 3.2 channels (IC 50 1.2 μ m ) [41].…”

“…This indicates that combination therapies or dual-acting peptides are worth pursuing. Whereas Pn3a has not yet been established as a dual inhibitor of other pain-relevant voltage-gated ion channels, a recent study demonstrated that the peptide inhibits Ca V 3.3 with low potency [13]. However, the implication of this target in the nociceptive pathway is less supported in the literature [64,69].…”

“…Recently, μ‐theraphotoxin‐Pn3a, isolated from the venom of Pamphobeteus nigricolor was characterized as a Na V 1.7 sodium channel inhibitor. Interestingly, a recent study demonstrated that Pn3a also inhibited the Ca V 1 and Ca V 2 subtype families, as well as Ca V 3.3 channels at high concentrations [12,13]. Interestingly, when administered in combination with subtherapeutic doses of opioids, the Pn3a peptide can produce profound analgesic activity in inflammatory animal models [14].…”