Dataset Information

Differential binding of tetrodotoxin and its derivatives to voltage-sensitive sodium channel subtypes (Na_v 1.1 to Na_v 1.7).

ABSTRACT:

Background and purpose

The development of subtype-selective ligands to inhibit voltage-sensitive sodium channels (VSSCs) has been attempted with the aim of developing therapeutic compounds. Tetrodotoxin (TTX) is a toxin from pufferfish that strongly inhibits VSSCs. Many TTX analogues have been identified from marine and terrestrial sources, although their specificity for particular VSSC subtypes has not been investigated. Herein, we describe the binding of 11 TTX analogues to human VSSC subtypes Na_v 1.1-Na_v 1.7.

Experimental approach

Each VSSC subtype was transiently expressed in HEK293T cells. The inhibitory effects of TTX analogues on each subtype were assessed using whole-cell patch-clamp recordings.

Key results

The inhibitory effects of TTX on Na_v 1.1-Na_v 1.7 were observed in accordance with those reported in the literature; however, the 5-deoxy-10,7-lactone-type analogues and 4,9-anhydro-type analogues did not cause inhibition. Chiriquitoxin showed less binding to Na_v 1.7 compared to the other TTX-sensitive subtypes. Two amino acid residues in the TTX binding site of Na_v 1.7, Thr¹⁴²⁵ and Ile¹⁴²⁶ were mutated to Met and Asp, respectively, because these residues were found at the same positions in other subtypes. The two mutants, Na_v 1.7 T1425M and Na_v 1.7 I1426D, had a 16-fold and 5-fold increase in binding affinity for chiriquitoxin, respectively.

Conclusions and implications

The reduced binding of chiriquitoxin to Na_v 1.7 was attributed to its C11-OH and/or C12-NH₂ , based on reported models for the TTX-VSSC complex. Chiriquitoxin is a useful tool for probing the configuration of the TTX binding site until a crystal structure for the mammalian VSSC is solved.

SUBMITTER: Tsukamoto T

PROVIDER: S-EPMC5647187 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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Publications

Differential binding of tetrodotoxin and its derivatives to voltage-sensitive sodium channel subtypes (Na<sub>v</sub> 1.1 to Na<sub>v</sub> 1.7).

Tsukamoto Tadaaki T Chiba Yukie Y Wakamori Minoru M Yamada Tomoshi T Tsunogae Shunsuke S Cho Yuko Y Sakakibara Ryo R Imazu Takuya T Tokoro Shouta S Satake Yoshiki Y Adachi Masaatsu M Nishikawa Toshio T Yotsu-Yamashita Mari M Konoki Keiichi K

British journal of pharmacology 20170920 21

<h4>Background and purpose</h4>The development of subtype-selective ligands to inhibit voltage-sensitive sodium channels (VSSCs) has been attempted with the aim of developing therapeutic compounds. Tetrodotoxin (TTX) is a toxin from pufferfish that strongly inhibits VSSCs. Many TTX analogues have been identified from marine and terrestrial sources, although their specificity for particular VSSC subtypes has not been investigated. Herein, we describe the binding of 11 TTX analogues to human VSSC ...[more]

PMID: 28832970

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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

Differential binding of tetrodotoxin and its derivatives to voltage-sensitive sodium channel subtypes (Na_v 1.1 to Na_v 1.7).

Background and purpose

Experimental approach

Key results

Conclusions and implications

Publications

Differential binding of tetrodotoxin and its derivatives to voltage-sensitive sodium channel subtypes (Na<sub>v</sub> 1.1 to Na<sub>v</sub> 1.7).

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Tweets

Dataset Information

Differential binding of tetrodotoxin and its derivatives to voltage-sensitive sodium channel subtypes (Nav 1.1 to Nav 1.7).

Background and purpose

Experimental approach

Key results

Conclusions and implications

Publications

Differential binding of tetrodotoxin and its derivatives to voltage-sensitive sodium channel subtypes (Na<sub>v</sub> 1.1 to Na<sub>v</sub> 1.7).

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Differential binding of tetrodotoxin and its derivatives to voltage-sensitive sodium channel subtypes (Na_v 1.1 to Na_v 1.7).