Effects of 4,9-anhydrotetrodotoxin on voltage-gated Na+ channels of mouse vas deferens myocytes and recombinant NaV1.6 channels
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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.
Keywords4,9-Anhydrotetrodotoxin β1-subunit NaV1.6 channels Smooth muscle-type NaV Mouse vas deferens
Analysis of variance
Fetal bovine serum
Green fluorescent protein
- HEK293 cells
Human embryonic kidney 293 cells
Inner circular muscle
Voltage-gated Na+ currents
- I-V relationships
Liquid chromatography-fluorescent detection
Motor end-plate disease
- NaV channels
Voltage-gated Na+ channels
Optimal cutting temperature
Outer longitudinal muscle
Phosphate buffered saline
Physiological salt solution
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.
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