N-linked glycosylation of ether á go-go potassium channels: effects on cell surface expression and functional properties

Ether a go-go (Eag1) is a voltage gated potassium channel, apparently involved in a broad variety of cellular events. Results from this work show that in rat tissues the Eag1 protein is predominantly expressed in brain tissue where it undergoes N-linked glycosylation, a cotranslational modification known to affect biogenesis and functional properties of numerous plasma membrane proteins. Analogously, human Eag1 undergoes N-linked glycosylation when expressed in heterologous systems.The human Eag1, as expressed in CHO cells, and the rat Eag1 natively expressed in brain, each exist as two isoforms, which harbor different asparagine-linked oligosaccharides. Examination of the carbohydrate attachments with endoglycosidases revealed two distinct complexes, the core-oligosaccharide and the complex-oligosaccharide, which contribute to the molecular masses of the hEag1 isoforms with 2-5 kDa and 20-25 kDa, respectively. Mutation of the asparagine residues in two of the six putative N-linked glycosylation motifs (N-X-S/T), Asn 388 and Asn406 altered the glycosylation of hEag1 channels. However, single amino acid exchanges in the four remaining glycosylation motifs had no effect on hEag1 glycosylation.Asparagine 388 is the site of attachment of the core-oligosaccharide, which is sensitive to Endoglycosidase H and recognisable by Concanavalin A. Mutation of this site had no significant effect on the intracellular localisation and functional properties of hEag1 channels, although interference with some other unidentified function cannot be excluded. Asparagine 406 carries the complex-oligosaccharide, which is resistant to Endoglycosidase H and unrecognisable by Concanavalin A. The attachment of the complex-oligosaccharide seems to have an important and complex function since mutation of the Asn 406 results in an altered phenotype. Intracellular distribution of hEag1 lacking the complex-oligosaccharide is strongly affected, showing a complex-perinuclear localisation instead of the widespread staining pattern. This is the case also for the hEag1 mutant lacking both glycosylation sites. Immunofluorescence experiments indicate that channel prot eins lacking the complex-oligosaccharides are, at least partially, trapped in the endoplasmatic reticulum. This can be explained, e.g., by folding problems due to the loss of oligosaccharide attachment. The altered intracellular localisation of the unglycosylated and the solely core-glycosylated protein is not likely due to the introduced mutation, since the same effect was obtained by expression of the wild-type protein in glycosylation deficient cells. Although the unglycosylated protein is partially trapped in the endoplasmatic reticulum, significant populations of mutant channels must be expressed on the cell surface, since hEag1 potassium currents are also detectable in cells expressing unglycosylated channels, albeit with a strongly reduced amplitude. Loss of the core -oligosaccharide only has no significant effect on hEag1 current amplitude.Furthermore, loss of the complex-oligosaccharide results in a shift of the voltage-dependence towards more positive potentials, a phenomenon commonly observed in the case of desialylation of voltage-dependent sodium channels. Additionally PIAS1 was identified as a putative hEag1 interaction partner of the C-terminus of hEag1 by two independent Yeast-Two-Hybrid screens. PIAS1 is expressed in nuclei where it acts as inhibitor of STAT mediated gene-activation. The hypothesis that a membrane protein, or at least part of it, can functionally interact with a nuclear protein is controversial, although recently evidence for such interactions are accumulating. This is of particular interest for Eag1, given its involvement in cancer.