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Mapping the Molecular Surface of the Analgesic Na_V1.7-Selective Peptide Pn3a Reveals Residues Essential for Membrane and Channel Interactions.

ABSTRACT: Compelling human genetic studies have identified the voltage-gated sodium channel Na_V1.7 as a promising therapeutic target for the treatment of pain. The analgesic spider-venom-derived peptide ?-theraphotoxin-Pn3a is an exceptionally potent and selective inhibitor of Na_V1.7; however, little is known about the structure-activity relationships or channel interactions that define this activity. We rationally designed 17 Pn3a analogues and determined their activity at hNa_V1.7 using patch-clamp electrophysiology. The positively charged amino acids K22 and K24 were identified as crucial for Pn3a activity, with molecular modeling identifying interactions of these residues with the S3-S4 loop of domain II of hNa_V1.7. Removal of hydrophobic residues Y4, Y27, and W30 led to a loss of potency (>250-fold), while replacement of negatively charged D1 and D8 residues with a positively charged lysine led to increased potencies (>13-fold), likely through alterations in membrane lipid interactions. Mutating D8 to an asparagine led to the greatest improvement in Pn3a potency at Na_V1.7 (20-fold), while maintaining >100-fold selectivity over the major off-targets Na_V1.4, Na_V1.5, and Na_V1.6. The Pn3a[D8N] mutant retained analgesic activity in vivo, significantly attenuating mechanical allodynia in a clinically relevant mouse model of postsurgical pain at doses 3-fold lower than those with wild-type Pn3a, without causing motor-adverse effects. Results from this study will facilitate future rational design of potent and selective peptidic Na_V1.7 inhibitors for the development of more efficacious and safer analgesics as well as to further investigate the involvement of Na_V1.7 in pain.

SUBMITTER: Mueller A

PROVIDER: S-EPMC7296542 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Mapping the Molecular Surface of the Analgesic NaV1.7-Selective Peptide Pn3a Reveals Residues Essential for Membrane and Channel Interactions.

Mueller Alexander A Dekan Zoltan Z Kaas Quentin Q Agwa Akello J AJ Starobova Hana H Alewood Paul F PF Schroeder Christina I CI Mobli Mehdi M Deuis Jennifer R JR Vetter Irina I

ACS pharmacology & translational science 20200219 3

Compelling human genetic studies have identified the voltage-gated sodium channel NaV1.7 as a promising therapeutic target for the treatment of pain. The analgesic spider-venom-derived peptide μ-theraphotoxin-Pn3a is an exceptionally potent and selective inhibitor of NaV1.7; however, little is known about the structure-activity relationships or channel interactions that define this activity. We rationally designed 17 Pn3a analogues and determined their activity at hNaV ...[more]

PMID: 32566918

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Project description:The human TE671 cell line was originally used as a model of medulloblastoma but has since been reassigned as rhabdomyosarcoma. Despite the characterised endogenous expression of voltage-sensitive sodium currents in these cells, the specific voltage-gated sodium channel (VGSC) subtype underlying these currents remains unknown. To profile the VGSC subtype in undifferentiated TE671 cells, endpoint and quantitative reverse transcription-PCR (qRT-PCR), western blot and whole-cell patch clamp electrophysiology were performed. qRT-PCR profiling revealed that expression of the SCN9A gene was ∼215-fold greater than the SCN4A gene and over 400-fold greater than any of the other VGSC genes, while western blot confirmed that the dominant SCN9A RNA was translated to a protein with a molecular mass of ∼250 kDa. Elicited sodium currents had a mean amplitude of 2.6 ± 0.7 nA with activation and fast inactivation V50 values of -31.9 ± 1.1 and -69.6 ± 1.0 mV, respectively. The currents were completely and reversibly blocked by tetrodotoxin at concentrations greater than 100 nm (IC50 = 22.3 nm). They were also very susceptible to the NaV 1.7 specific blockers Huwentoxin-IV and Protoxin-II with IC50 values of 14.6 nm and 0.8 nm, respectively, characteristic of those previously determined for NaV 1.7. Combined, the results revealed the non-canonical and highly dominant expression of NaV 1.7 in the human TE671 rhabdomyosarcoma cell line. We show that the TE671 cell line is an easy to maintain and cost-effective model for the study of NaV 1.7, a major target for the development of analgesic drugs and more generally for the study of pain. KEY POINTS: Undifferentiated TE671 cells produce a voltage-sensitive sodium current when depolarised. The voltage-gated sodium channel isoform expressed in undifferentiated TE671 cells was previously unknown. Through qRT-PCR, western blot and toxin pharmacology, it is shown that undifferentiated TE671 cells dominantly (>99.5%) express the NaV 1.7 isoform that is strongly associated with pain. The TE671 cell line is, therefore, a very easy to maintain and cost-effective model to study NaV 1.7-targeting drugs.

Dataset Information

Mapping the Molecular Surface of the Analgesic Na_V1.7-Selective Peptide Pn3a Reveals Residues Essential for Membrane and Channel Interactions.

Publications

Mapping the Molecular Surface of the Analgesic Na<sub>V</sub>1.7-Selective Peptide Pn3a Reveals Residues Essential for Membrane and Channel Interactions.

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