title: Unconventional myristoylation of large-conductance Ca²⁺-activated K⁺ channel (Slo1) via serine/threonine residues regulates channel surface expression.
abstract: Protein myristoylation is a means by which cells anchor proteins into membranes. The most common type of myristoylation occurs at an N-terminal glycine. However, myristoylation rarely occurs at an internal amino acid residue. Here we tested whether the α-subunit of the human large-conductance voltage- and Ca(2+)-activated K(+) channel (hSlo1) might undergo internal myristoylation. hSlo1 expressed in HEK293T cells incorporated [(3)H]myristic acid via a posttranslational mechanism, which is insensitive to cycloheximide, an inhibitor of protein biosynthesis. In-gel hydrolysis of [(3)H]myristoyl-hSlo1 with alkaline NH(2)OH (which cleaves hydroxyesters) but not neutral NH(2)OH (which cleaves thioesters) completely removed [(3)H]myristate from hSlo1, suggesting the involvement of a hydroxyester bond between hSlo1's hydroxyl-bearing serine, threonine, and/or tyrosine residues and myristic acid; this type of esterification was further confirmed by its resistance to alkaline Tris·HCl. Treatment of cells expressing hSlo1 with 100 μM myristic acid caused alteration of hSlo1 activation kinetics and a 40% decrease in hSlo1 current density from 20 to 12 nA*MΩ. Immunocytochemistry confirmed a decrease in hSlo1 plasmalemma localization by myristic acid. Replacement of the six serines or the seven threonines (but not of the single tyrosine) of hSlo1 intracellular loops 1 and 3 with alanines decreased hSlo1 direct myristoylation by 40-44%, whereas in combination decreased myristoylation by nearly 90% and abolished the myristic acid-induced change in current density. Our data demonstrate that an ion channel, hSlo1, is internally and posttranslationally myristoylated. Myristoylation occurs mainly at hSlo1 intracellular loop 1 or 3, and is an additional mechanism for channel surface expression regulation.
authors.affiliation: Division of Molecular Medicine, Department of Anesthesiology, University of California, Los Angeles, CA 90095-1778, USA.
authors.forename: Abderrahmane
authors.initials: A
authors.lastname: Alioua
authors.forename: Min
authors.initials: M
authors.lastname: Li
authors.forename: Yong
authors.initials: Y
authors.lastname: Wu
authors.forename: Enrico
authors.initials: E
authors.lastname: Stefani
authors.forename: Ligia
authors.initials: L
authors.lastname: Toro
mesh_terms: D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004952:Esters; D006801:Humans; D007150:Immunohistochemistry; D007700:Kinetics; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D019814:Myristic Acid / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D011499:Protein Processing, Post-Translational; D012694:Serine / Q000378:metabolism*; D013912:Threonine / Q000737:chemistry / Q000378:metabolism*
chemical_list: D004952:Esters; C494841:KCNMA1 protein, human; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D019814:Myristic Acid; D013912:Threonine; D012694:Serine