µ-Theraphotoxin Pn3a inhibition of CaV3.3 channels reveals a novel isoform-selective drug binding site
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Evaluation Summary:
Low voltage-activated T-type calcium channels (CaV3.1-3.3) are important for several physiological processes. It is challenging to distinguish their specific physiological / pathophysiological roles as they share similar biophysical properties, expression profiles and there is a lack of subtype selective pharmacology. This study reports a spider toxin, Pn3a, which exhibits 100-fold selectivity for inhibiting CaV3.3 over CaV3.1 and CaV3.2 isoforms, and which therefore makes for an excellent reagent for the physiological study of CaV3.3.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
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.
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Evaluation Summary:
Low voltage-activated T-type calcium channels (CaV3.1-3.3) are important for several physiological processes. It is challenging to distinguish their specific physiological / pathophysiological roles as they share similar biophysical properties, expression profiles and there is a lack of subtype selective pharmacology. This study reports a spider toxin, Pn3a, which exhibits 100-fold selectivity for inhibiting CaV3.3 over CaV3.1 and CaV3.2 isoforms, and which therefore makes for an excellent reagent for the physiological study of CaV3.3.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
Overall, the selectivity of Pn3a for CaV3.3 over CaV3.1 and CaV3.2 is convincingly demonstrated by the data presented. However, the lack of any experiments in native cells expressing a mixture of CaV3 channel isoforms is a significant omission in the study. Moreover, previous findings cited in the manuscript that Pn3a also inhibits Nav1.7 and high-voltage-activated (HVA) calcium channels further casts doubts as to how useful it would be in specifically dissecting effects of CaV3.3 in native tissues. Other weaknesses in the manuscript pertain to the lack of modeling to understand the mechanism of action of Pn3a on CaV3.3, and absence of experimental evidence to validate the computational docking model.
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Reviewer #2 (Public Review):
The low voltage activated T-type calcium channels (CaV3.1-3.3) play a wide range of physiological roles, yet discerning their functions is challenging as options for selective pharmacology is limited. Although there are toxins such as ProTx-I and ProTx-II that target CaV3.1 and CaV3.2 with some specificity, there are no such tool available for CaV3.3. This study identifies the first subtype modulator of CaV3.3, which is interesting and will undoubtedly open new experimental avenues. The strength of the study is the extensive electrophysiological characterization of Pn3a modulation CaV3.1-3 channels, which is compelling. The weakness of the study is that the mechanisms that allow selective block of CaV3.3 is not fully clear.
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Reviewer #3 (Public Review):
The authors show that the sodium channel blocking spider toxin Pn3a inhibits Cav3.3 channels selectively over Cav3.2 and Cav3.2. The inhibitory effect has the hallmarks of a classical gating modifier like agaIVA or grammotoxin, and by using chimeras with a K channel and molecular docking analysis, they identify the voltage sensing domain of DII as the target. Nice ephys overall, but some points should be addressed. My main concern relates to the use of a chimeric K channel for this work - I am not sure that I agree with the author's statement that a calcium channel chimera would not have worked. To what extent this work will lead to new tools for making a Cav3.3 channel selective inhibitor remains to be seen.
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