Restoration of Inactivation in Mutants of <i>Shaker</i> Potassium Channels by a Peptide Derived from ShB

TLDR

Site‑directed mutagenesis of Drosophila Shaker potassium channels suggests that the first 20 cytoplasmic residues form a domain that interacts with the open channel to trigger inactivation. The hypothesis was tested by internally applying a synthetic peptide comprising the first 20 residues of the ShB splice variant to noninactivating mutant channels expressed in Xenopus oocytes. The peptide restored inactivation in a concentration‑dependent, voltage‑independent manner, whereas trypsin‑treated or mutant‑derived peptides failed, confirming that a cytoplasmic domain occludes the pore to mediate inactivation.

Abstract

Site-directed mutagenesis experiments have suggested a model for the inactivation mechanism of Shaker potassium channels from Drosophila melanogaster. In this model, the first 20 amino acids form a cytoplasmic domain that interacts with the open channel to cause inactivation. The model was tested by the internal application of a synthetic peptide, with the sequence of the first 20 residues of the ShB alternatively spliced variant, to noninactivating mutant channels expressed in Xenopus oocytes. The peptide restored inactivation in a concentration-dependent manner. Like normal inactivation, peptide-induced inactivation was not noticeably voltage-dependent. Trypsin-treated peptide and peptides with sequences derived from the first 20 residues of noninactivating mutants did not restore inactivation. These results support the proposal that inactivation occurs by a cytoplasmic domain that occludes the ion-conducting pore of the channel.