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, Lys(27). By probing the pore of the Kv1.3 channel with synthetic kaliotoxin-Lys(27) mutants, each containing a non-natural lysine analog of a different length, and using mutant cycle analysis, we determined the spatial locations of Tyr(400) and Asp(402) in the GYGD moth, relative to His(404) located at the base of the outer vestibule. Our data indicate that the terminal amines of the shorter Lys(27) analogs lie close to His(404) and to Asp(402) while Lys(27) itself interacts with Tyr(400). 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 (similar to 8-9-Angstrom diameter) formed from alternating Asp(402) and His(404) residues. Tyr(400) lies 4-6 Angstrom deeper into the pore, and its interaction with kaliotoxin-Lys(27) is in competition with K+ ions, Studies with dimeric Kv1.3 constructs suggest that two Tyr(400) 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.