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Address correspondence to Kenton J. Swartz, Molecular Physiology and Biophysics Unit, National Institute of Neurological Disorders and Stroke/National Institutes of Health, Building 36, Room 2C19 36, Convent Dr., MSC 4066, Bethesda, MD 20892. Fax: (301) 435-5666; E-mail: swartzk{at}ninds.nih.gov
In Kv channels, an activation gate is thought to be located near the intracellular entrance to the ion conduction pore. Although the COOH terminus of the S6 segment has been implicated in forming the gate structure, the residues positioned at the occluding part of the gate remain undetermined. We use a mutagenic scanning approach in the Shaker Kv channel, mutating each residue in the S6 gate region (T469-Y485) to alanine, tryptophan, and aspartate to identify positions that are insensitive to mutation and to find mutants that disrupt the gate. Most mutants open in a steeply voltage-dependent manner and close effectively at negative voltages, indicating that the gate structure can both support ion flux when open and prevent it when closed. We find several mutant channels where macroscopic ionic currents are either very small or undetectable, and one mutant that displays constitutive currents at negative voltages. Collective examination of the three types of substitutions support the notion that the intracellular portion of S6 forms an activation gate and identifies V478 and F481 as candidates for occlusion of the pore in the closed state.
Key Words: Kv channel • voltage-dependent gating • scanning mutagenesis • pore occlusion • closed gate
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