Elucidating the roles of voltage sensors in NaV1.9 activation and inactivation through a spider toxin.

The gating process of voltage-gated sodium (NaV) channels is extraordinary intrinsic and involves numerous factors, such as voltage-sensing domain (VSD), the N-terminus and C-terminus, and the auxiliary subunits. To date, the gating mechanism of NaV channel has not been clearly elucidated. NaV1.9 has garnered significant attention due to its slow gating kinetics. Due to the challenges of NaV1.9 heterologous expression, research on its gating mechanism is relatively limited. Whether there are any differences in the functions of the four VSDs in NaV1.9 compared to those in other subtypes remains an open question. Here, we employed the established chimera method to transplant the S3b-S4 motif from the VSDIV of the toxin-sensitive donor channel (NaV1.9) into the receptor channel (NaV1.9/1.8 DIV S3b-S4 chimera). This modification imparted animal toxin sensitivity to the other three VSDs. Our results demonstrate that all four VSDs of NaV1.9 are involved in channel opening, VSDIII and VSDIV are primarily involved in regulating fast inactivation, and VSDII also regulates the steady-state inactivation of channels. These findings provide a new insight into the gating mechanism of NaV1.9.