Abstract

Shaker Kv channels inactivate rapidly to culminate the action potential and maintain the homeostasis of excitable cells. The so-called N-type inactivation is caused by the first 46 amino acids of the N-terminus of the channel, known as the inactivation peptide. Numerous mutational studies have characterized N-type inactivation functionally, however, the position of the inactivation peptide in the resting state and its transition during inactivation is still debated. Here, we tracked the movement of the inactivation peptide during inactivation using voltage clamp fluorometry. By inserting an unnatural amino acid, 3-[(6-acetyl-2-naphthalenyl) amino]-L-alanine (Anap), which is sensitive to changes in environment, we identified the movements of ball and chain separately. Our data suggests that N-type inactivation occurs in a biphasic movement by first releasing the IP, which then blocks the pore from the cytoplasmic side. To further narrow down the resting position of the inactivation peptide, we used Lanthanide-based Resonance Energy transfer and transition metal (tm)FRET. We propose that the inactivation peptide is located in the window formed by the channel and the T1 domain, interacting with the acidic residues of the T1 domain.