Effects of 4,9-anhydrotetrodotoxin on voltage-gated Na + channels of mouse vas deferens myocytes and recombinant Na V 1.6 channels.

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 NaV 1.6 channels (expressed in HEK293 cells). A depolarizing rectangular voltage-pulse elicited a fast and transient inward native INa and recombinant NaV 1.6 expressed in HEK293 cells (i.e., recombinant INa ). The current decay of native INa was similar to the recombinant NaV 1.6 current co-expressed with β1 -subunits. The current-voltage (I-V) relationships of native INa were similar to those of recombinant NaV 1.6 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.