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

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 = 510nM), recombinant INa (K i = 112nM), and recombinant INa co-expressed with β1-subunits (K i = 92nM). 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.