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Effects of 4,9-anhydrotetrodotoxin on voltage-gated Na+ channels of mouse vas deferens myocytes and recombinant NaV1.6 channels

  • Kohei Takahara
  • Tadashi Yamamoto
  • Keiichiro Uchida
  • Hai-Lei Zhu
  • Atsushi Shibata
  • Tetsuichiro Inai
  • Mitsuru Noguchi
  • Mari Yotsu-Yamashita
  • Noriyoshi Teramoto
Original Article
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Abstract

Molecular investigations were performed in order to determine the major characteristics of voltage-gated Na+ channel β-subunits in mouse vas deferens. The use of real-time quantitative PCR showed that the expression of Scn1b was significantly higher than that of other β-subunit genes (Scn2b – Scn4b). Immunoreactivity of Scn1b proteins was also detected in the inner circular and outer longitudinal smooth muscle of mouse vas deferens. In whole-cell recordings, the actions of 4,9-anhydroTTX on voltage-gated Na+ current peak amplitude in myocytes (i.e., native INa) were compared with its inhibitory potency on recombinant NaV1.6 channels (expressed in HEK293 cells). A depolarizing rectangular voltage-pulse elicited a fast and transient inward native INa and recombinant NaV1.6 expressed in HEK293 cells (i.e., recombinant INa). The current decay of native INa was similar to the recombinant NaV1.6 current co-expressed with β1-subunits. The current-voltage (I-V) relationships of native INa were similar to those of recombinant NaV1.6 currents co-expressed with β1-subunits. Application of 4,9-anhydroTTX inhibited the peak amplitude of native INa (K i  = 510 nM), recombinant INa (K i  = 112 nM), and recombinant INa co-expressed with β1-subunits (K i  = 92 nM). The half-maximal (Vhalf) activation and inactivation of native INa values were similar to those observed in recombinant INa co-expressed with β1-subunits. These results suggest that β1-subunit proteins are likely to be expressed mainly in the smooth muscle layers of murine vas deferens and that 4,9-anhydroTTX inhibited not only native INa but also recombinant INa and recombinant INa co-expressed with β1-subunits in a concentration-dependent manner.

Keywords

4,9-Anhydrotetrodotoxin β1-subunit NaV1.6 channels Smooth muscle-type NaV Mouse vas deferens 

Abbreviations

4,9-anhydroTTX

4,9-Anhydrotetrodotoxin

ANOVA

Analysis of variance

FBS

Fetal bovine serum

GFP

Green fluorescent protein

HEK293 cells

Human embryonic kidney 293 cells

ICM

Inner circular muscle

INa

Voltage-gated Na+ currents

I-V relationships

Current-voltage relationships

LC-FLD

Liquid chromatography-fluorescent detection

med

Motor end-plate disease

NaV channels

Voltage-gated Na+ channels

OCT

Optimal cutting temperature

OLM

Outer longitudinal muscle

PBS

Phosphate buffered saline

PFA

Paraformaldehyde

PSS

Physiological salt solution

TEA

Tetraethylammonium

TTX

Tetrodotoxin

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Notes

Acknowledgments

The authors wish to thank Prof. Jiro Uozumi (Saga University, Department of Urology, Saga, Japan) for the helpful discussion and critical reading of the manuscript.

Funding

Japanese Society for the Promotion of Science (JSPS), Grant-in-Aid for Scientific Research (B); Grant Number: 17H02111.

JSPS, Grant-in-Aid for Challenging Exploratory Research; Grant Number: 16K12913.

JSPS, Grant-in-Aid for Young Scientists (B); Grant Number: 16K19020.

Compliance with ethical standards

The experimental protocols were approved by the Ethics Committee of Saga University, Saga, Japan.

Conflict of interest

The authors declare that they have no conflict of interests.

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Kohei Takahara
    • 1
  • Tadashi Yamamoto
    • 1
  • Keiichiro Uchida
    • 1
  • Hai-Lei Zhu
    • 1
  • Atsushi Shibata
    • 1
  • Tetsuichiro Inai
    • 2
  • Mitsuru Noguchi
    • 3
  • Mari Yotsu-Yamashita
    • 4
  • Noriyoshi Teramoto
    • 1
    • 5
  1. 1.Department of Pharmacology, Faculty of MedicineSaga UniversitySagaJapan
  2. 2.Department of Morphological Biology, Division of Biomedical SciencesFukuoka Dental CollegeFukuokaJapan
  3. 3.Department of Urology, Faculty of MedicineSaga UniversitySagaJapan
  4. 4.Laboratory of Bioorganic Chemistry of Natural Products, Graduate School of Agricultural ScienceTohoku UniversitySendaiJapan
  5. 5.Laboratory of Biomedical Engineering, Graduate School of Biomedical EngineeringTohoku UniversitySendaiJapan

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