Disulfide Locking Reveals Interaction of the Gating Charges with a Negative Charge in the S1 Segment During Activation of the NaChBac Voltage Sensor

Abstract

Opening and closing the pore of voltage-gated ion channels are mechanically linked to conformational movement of the positively charged fourth transmembrane segment (S4) in the voltage sensor. Disulfide locking of cysteines substituted for gating charges and for E43 at the extracellular end of the S1 segment of the bacterial sodium channel NaChBac demonstrated that a threonine at gating charge position 0 (T0) and arginine at the first gating charge position (R1) located at the extracellular end of S4 interact with E43 in the closed state, whereas the second and the third gating charges (R2) and (R3) interact with E43 in activated states. The kinetics and voltage dependence of disulfide locking of R2C and R3C demonstrate sequential interactions of the gating charges with E43C during voltage sensor activation. We did not observe disulfide locking with the fourth or last gating charge (R4) and E43, suggesting that we have described the upper limit to the outward movement made by the S4 during channel activation. These results indicate that S4 moves 8-10A outward with respect to E43 in the transition from resting to activate states, which supports a sliding helix model of voltage sensor movement in voltage-gated ion channels.