Genetic studies of sup-9, unc-93, and sup-10 strongly suggest that these genes encode components of a multi-subunit protein complex that coordinates muscle contraction in Caenorhabditis elegans. We cloned sup-9 and sup-10 and found that they encode a two-pore K ϩ channel and a novel transmembrane protein, respectively. We also found that UNC-93 and SUP-10 colocalize with SUP-9 within muscle cells, and that UNC-93 is a member of a novel multigene family that is conserved among C. elegans, Drosophila, and humans. Our results indicate that SUP-9 and perhaps other two-pore K ϩ channels function as multiprotein complexes, and that UNC-93 and SUP-10 likely define new classes of ion channel regulatory proteins.
Radiolabeling experiments in AtT 20 cells in the presence of the drug brefeldin A, which blocks transport from the endoplasmic reticulum to the Golgi complex, led to an accumulation of the 26-kDa precursor, suggesting a post-endoplasmic reticulum site of processing. When Golgi complex-to-secretory granule transport was blocked at 20°C, the processing of the 26-kDa precursor was not affected, suggesting a Golgi complex site of processing. At this temperature, the 15-kDa N-terminal intermediate accumulated, suggesting a post-Golgi complex processing site, while the 16.5-kDa C-terminal intermediate was processed in the Golgi complex to produce a 5.4-kDa peptide.
Loss-of-function mutations in the Caenorhabditis elegans gene sup-18 suppress the defects in muscle contraction conferred by a gain-of-function mutation in SUP-10, a presumptive regulatory subunit of the SUP-9 two-pore domain K+ channel associated with muscle membranes. We cloned sup-18 and found that it encodes the C. elegans ortholog of mammalian iodotyrosine deiodinase (IYD), an NADH oxidase/flavin reductase that functions in iodine recycling and is important for the biosynthesis of thyroid hormones that regulate metabolism. The FMN-binding site of mammalian IYD is conserved in SUP-18, which appears to require catalytic activity to function. Genetic analyses suggest that SUP-10 can function with SUP-18 to activate SUP-9 through a pathway that is independent of the presumptive SUP-9 regulatory subunit UNC-93. We identified a novel evolutionarily conserved serine-cysteine-rich region in the C-terminal cytoplasmic domain of SUP-9 required for its specific activation by SUP-10 and SUP-18 but not by UNC-93. Since two-pore domain K+ channels regulate the resting membrane potentials of numerous cell types, we suggest that the SUP-18 IYD regulates the activity of the SUP-9 channel using NADH as a coenzyme and thus couples the metabolic state of muscle cells to muscle membrane excitability.
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