Unconventional myristoylation of large-conductance Ca²⁺-activated K⁺ channel (Slo1) via serine/threonine residues regulates channel surface expression.

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.