The signature sequence of voltage-gated potassium channels projects into the external vestibule.

A highly conserved motif, GYGD, contributes to the formation of the ion selectivity filter in voltage-gated K+ channels and is thought to interact with the scorpion toxin residue, Lys27. By probing the pore of the Kv1.3 channel with synthetic kaliotoxin-Lys27 mutants, each containing a non-natural lysine analog of a different length, and using mutant cycle analysis, we determined the spatial locations of Tyr400 and Asp402 in the GYGD motif, relative to His404 located at the base of the outer vestibule. Our data indicate that the terminal amines of the shorter Lys27 analogs lie close to His404 and to Asp402, while Lys27 itself interacts with Tyr400. Based on these data, we developed a molecular model of this region of the channel. The junction between the outer vestibule and the pore is defined by a ring ( approximately 8-9-A diameter) formed from alternating Asp402 and His404 residues. Tyr400 lies 4-6 A deeper into the pore, and its interaction with kaliotoxin-Lys27 is in competition with K+ ions. Studies with dimeric Kv1.3 constructs suggest that two Tyr400 residues in the tetramer are sufficient to bind K+ ions. Thus, at least part of the K+ channel signature sequence extends into a shallow trough at the center of a wide external vestibule.