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
First Online:
Received:
Accepted:
  • 102 Downloads

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

This is a preview of subscription content, log in to check access

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.

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.

References

  1. Brackenbury WJ, Calhoun JD, Chen C, Miyazaki H, Nukina N, Oyama F, Ranscht B, Isom LL (2010) Functional reciprocity between Na+ channel NaV1.6 and beta1 subunits in the coordinated regulation of excitability and neurite outgrowth. Proc Natl Acad Sci U S A 107(5):2283–2288.  https://doi.org/10.1073/pnas.0909434107 CrossRefPubMedPubMedCentralGoogle Scholar
  2. Catterall WA, Goldin AL, Waxman SG (2005) International union of pharmacology. XLVII. Nomenclature and structure-function relationships of voltage-gated sodium channels. Pharmacol Rev 57(4):397–409.  https://doi.org/10.1124/pr.57.4.4 CrossRefPubMedGoogle Scholar
  3. Clare JJ, Tate SN, Nobbs M, Romanos MA (2000) Voltage-gated sodium channels as therapeutic targets. Drug Discov Today 5(11):506–520.  https://doi.org/10.1016/S1359-6446(00)01570-1 CrossRefPubMedGoogle Scholar
  4. Ferrera L, Moran O (2006) β1-subunit modulates the NaV1.4 sodium channel by changing the surface charge. Exp Brain Res 172(2):139–150.  https://doi.org/10.1007/s00221-005-0323-4 CrossRefPubMedGoogle Scholar
  5. Goldin AL (2001) Resurgence of sodium channel research. Annu Rev Physiol 63(1):871–894.  https://doi.org/10.1146/annurev.physiol.63.1.871 CrossRefPubMedGoogle Scholar
  6. Kazarinova-Noyes K, Malhotra JD, McEwen DP, Mattei LN, Berglund EO, Ranscht B, Levinson SR, Schachner M, Shrager P, Isom LL, Xiao ZC (2001) Contactin associates with Na+ channels and increases their functional expression. J Neurosci 21(19):7517–7525PubMedGoogle Scholar
  7. Liu CJ, Dib-Hajj SD, Black JA, Greenwood J, Lian Z, Waxman SG (2001) Direct interaction with contactin targets voltage-gated sodium channel NaV1.9/NaN to the cell membrane. J Biol Chem 276(49):46553–46561.  https://doi.org/10.1074/jbc.M108699200 CrossRefPubMedGoogle Scholar
  8. Nakamura M, Yasumoto T (1985) Tetrodotoxin derivatives in puffer fish. Toxicon 23(2):271–276.  https://doi.org/10.1016/0041-0101(85)90149-7 CrossRefPubMedGoogle Scholar
  9. Rosker C, Lohberger B, Hofer D, Steinecker B, Quasthoff S, Schreibmayer W (2007) The TTX metabolite 4,9-anhydro-TTX is a highly specific blocker of the NaV1.6 voltage-dependent sodium channel. Am J Physiol Cell Physiol 293(2):C783–C789.  https://doi.org/10.1152/ajpcell.00070.2007 CrossRefPubMedGoogle Scholar
  10. Saleh S, Yeung SY, Prestwich S, Pucovský V, Greenwood I (2005) Electrophysiological and molecular identification of voltage-gated sodium channels in murine vascular myocytes. J Physiol 568(1):155–169.  https://doi.org/10.1113/jphysiol.2005.090951 CrossRefPubMedPubMedCentralGoogle Scholar
  11. Seda M, Pinto FM, Wray S, Cintado CG, Noheda P, Buschmann H, Candenas L (2007) Functional and molecular characterization of voltage-gated sodium channels in uteri from nonpregnant rats. Biol Reprod 77(5):855–863.  https://doi.org/10.1095/biolreprod.107.063016 CrossRefPubMedGoogle Scholar
  12. Teramoto N, Brading AF (1996) Activation by levcromakalim and metabolic inhibition of glibenclamide-sensitive K channels in smooth muscle cells of pig proximal urethra. Br J Pharmacol 118(3):635–642.  https://doi.org/10.1111/j.1476-5381.1996.tb15448.x CrossRefPubMedPubMedCentralGoogle Scholar
  13. Teramoto N, Yotsu-Yamashita M (2015) Selective blocking effects of 4,9-anhydrotetrodotoxin, purified from a crude mixture of tetrodotoxin analogues, on NaV1.6 channels and its chemical aspects. Marine Drugs 13(12):984–995.  https://doi.org/10.3390/md13020984 CrossRefPubMedPubMedCentralGoogle Scholar
  14. Teramoto N, Zhu HL, Yotsu-Yamashita M, Inai T, Cunnane TC (2012) Resurgent-like currents in mouse vas deferens myocytes are mediated by NaV1.6 voltage-gated sodium channels. Pflügers Arch Euro J Physiol 464(5):493–502.  https://doi.org/10.1007/s00424-012-1153-4 CrossRefGoogle Scholar
  15. Yotsu-Yamashita M, Goto A, Nakagawa T (2005) Identification of 4-S-Cysteinyltetrodotoxin from the liver of the puffer fish, Fugu pardalis, and formation of thiol adducts of tetrodotoxin from 4,9-anhydrotetrodotoxin. Chem Res Toxicol 18(5):865–871.  https://doi.org/10.1021/tx050015g CrossRefPubMedGoogle Scholar
  16. Zhao J, O’Leary ME, Chahine M (2011) Regulation of NaV1.6 and NaV1.8 peripheral nerve Na+ channels by auxiliary β-subunits. J Neurophysiol 106(2):608–619.  https://doi.org/10.1152/jn.00107.2011 CrossRefPubMedPubMedCentralGoogle Scholar
  17. Zhu HL, Aishima M, Morinaga H, Wassall RD, Shibata A, Iwasa K, Nomura M, Nagao M, Sueishi K, Cunnane TC, Teramoto N (2008) Molecular and biophysical properties of voltage-gated Na+ channels in murine vas deferens. Biophys J 94(8):3340–3351.  https://doi.org/10.1529/biophysj.107.117192 CrossRefPubMedPubMedCentralGoogle Scholar
  18. Zhu HL, Wassall RD, Takai M, Morinaga H, Nomura M, Cunnane TC, Teramoto N (2009) Actions of veratridine on tetrodotoxin-sensitive voltage-gated Na+ currents, NaV1.6, in murine vas deferens myocytes. Br J Pharmacol 157(8):1483–1493.  https://doi.org/10.1111/j.1476-5381.2009.00301.x CrossRefPubMedPubMedCentralGoogle Scholar
  19. Zhu HL, Shibata A, Inai T, Nomura M, Shibata Y, Brock JA, Teramoto N (2010) Characterization of NaV1.6-mediated Na+ currents in smooth muscle cells isolated from mouse vas deferens. J Cell Physiol 223(1):234–243.  https://doi.org/10.1002/jcp.22032 PubMedGoogle Scholar

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

Personalised recommendations