Step A shows channel opens after domains 1–3 activate; domain 4 moves, generating the slow gating component . Step B shows binding site exposed; IFM motif binds linker, following domain 4 movement and producing fast fluorescence signal during early inactivation steps. Step C shows IFM stabilizes via hydrogen bonds; domain 4 immobilizes, and S6 segments rotate, strengthening interactions and generating slower fluorescence signal changes. Step D shows hydrophobic gate forms as S6 rotates; pore closes, sodium current stops, and domain 3 voltage sensor becomes immobilized, completing inactivation.
States and events that lead to inactivation: the lock and key model. (A) This sequence starts after the VSDs of the first three domains have moved and the channel has opened, as indicated by the early part of the inward Na current (the lock is blocked). Then the VSD of DIV moves, leading to the slow component of the gating current. (B) The site in the S3–S4 linker of DIV is exposed, enabling the binding of the IFM motif. (The lock is exposed). The movement of IFM follows that of S4 of DIV, and it is shown as the fast ANAP signal recorded when ANAP replaces the F of the IFM. (C) IFM is bound via two pairs of H-bonds, producing the immobilization of the charge of domain IV. (Key is in the lock). With the IFM in position, a rotation of S6 segments of DIII and DIV stabilizes both S6 segments with the phenylalanine in DIII and the isoleucine in domain IV (in orange) by contacting the IFM. If ANAP replaces the F of IFM, this movement generates the slow component of the fluorescence signal. (D) The rotation has positioned in the pore all four hydrophobic residues (yellow circles) that form the inactivation gate, thus blocking conduction, shown as the inactivation of the sodium current. At the same time, the contact of the phenylalanine of S6 of DIII with the IFM immobilizes the VSD of DIII. (The lock has rotated and locked).