N-glycosylation promotes the cell surface expression of Kv1.3 potassium channels.
ABSTRACT The voltage-gated potassium channel Kv1.3 plays an essential role in modulating membrane excitability in many cell types. Kv1.3 is a heavily glycosylated membrane protein. Two successive N-glycosylation consensus sites, N228NS and N229ST, are present on the S1-S2 linker of rat Kv1.3. Our data suggest that Kv1.3 contains only one N-glycan and it is predominantly attached to N229 in the S1-S2 extracellular linker. Preventing N-glycosylation of Kv1.3 significantly decreased its surface protein level and surface conductance density level, which were ∼ 49% and ∼ 46% respectively of the level of wild type. Supplementation of N-acetylglucosamine (GlcNAc), l-fucose or N-acetylneuraminic acid to the culture medium promoted Kv1.3 surface protein expression, whereas supplementation of d-glucose, d-mannose or d-galactose did not. Among the three effective monosaccharides/derivatives, adding GlcNAc appeared to reduce sialic acid content and increase the degree of branching in the N-glycan of Kv1.3, suggesting that the N-glycan structure and composition had changed. Furthermore, the cell surface half-life of the Kv1.3 surface protein was increased upon GlcNAc supplementation, indicating that it had decreased internalization. The GlcNAc effect appears to apply mainly to membrane proteins containing complex type N-glycans. Thus, N-glycosylation promotes Kv1.3 cell surface expression; supplementation of GlcNAc increased Kv1.3 surface protein level and decreased its internalization, presumably by a combined effect of decreased branch size and increased branching of the N-glycan.
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ABSTRACT: Kv1.1 and Kv1.4 potassium channels are plasma membrane glycoproteins involved in action potential repolarization. We have shown previously that glycosylation affects the gating function of Kv1.1 and that a pore region determinant of Kv1.1 and Kv1.4 affects their cell surface trafficking negatively or positively, respectively. Here we investigated the role of N-glycosylation of Kv1.1 and Kv1.4 on their protein stability, cellular localization pattern, and trafficking to the cell surface. We found that preventing N-glycosylation of Kv1.4 decreased its protein stability, induced its high partial intracellular retention, and decreased its cell surface protein levels, whereas it had little or no effect on these parameters for Kv1.1. Exchanging a trafficking pore region determinant between Kv1.1 and Kv1.4 reversed these effects of glycosylation on these chimeric channels. Thus it appeared that the Kv1.4 pore region determinant and the sugar tree attached to the S1-S2 linker showed some type of dependence in promoting proper trafficking of the protein to the cell surface, and this dependence can be transferred to chimeric Kv1.1 proteins that contain the Kv1.4 pore. Understanding the different trafficking programs of Kv1 channels, and whether they are altered by glycosylation, will highlight the different posttranslational mechanisms available to cells to modify their cell surface ion channel levels and possibly their signaling characteristics.Journal of Biological Chemistry 04/2004; 279(10):8879-85. · 4.65 Impact Factor
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ABSTRACT: The lymphocyte K+ channel Kv1.3 constitutes an attractive pharmacological target for the selective suppression of terminally differentiated effector memory T (TEM) cells in T cell-mediated autoimmune diseases, such as multiple sclerosis and type 1 diabetes. Unfortunately, none of the existing small-molecule Kv1.3 blockers is selective, and many of them, such as correolide, 4-phenyl-4-[3-(methoxyphenyl)-3-oxo-2-azapropyl]cyclohexanone, and our own compound Psora-4 inhibit the cardiac K+ channel Kv1.5. By further exploring the structure-activity relationship around Psora-4 through a combination of traditional medicinal chemistry and whole-cell patch-clamp, we identified a series of new phenoxyalkoxypsoralens that exhibit 2- to 50-fold selectivity for Kv1.3 over Kv1.5, depending on their exact substitution pattern. The most potent and "drug-like" compound of this series, 5-(4-phenoxybutoxy)psoralen (PAP-1), blocks Kv1.3 in a use-dependent manner, with a Hill coefficient of 2 and an EC50 of 2 nM, by preferentially binding to the C-type inactivated state of the channel. PAP-1 is 23-fold selective over Kv1.5, 33- to 125-fold selective over other Kv1-family channels, and 500- to 7500-fold selective over Kv2.1, Kv3.1, Kv3.2, Kv4.2, HERG, calcium-activated K+ channels, Na+,Ca2+, and Cl- channels. PAP-1 does not exhibit cytotoxic or phototoxic effects, is negative in the Ames test, and affects cytochrome P450-dependent enzymes only at micromolar concentrations. PAP-1 potently inhibits the proliferation of human TEM cells and suppresses delayed type hypersensitivity, a TEM cell-mediated reaction, in rats. PAP-1 and several of its derivatives therefore constitute excellent new tools to further explore Kv1.3 as a target for immunosuppression and could potentially be developed into orally available immunomodulators.Molecular Pharmacology 12/2005; 68(5):1254-70. · 4.41 Impact Factor
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ABSTRACT: Asparagine-linked glycosylation (N-glycosylation) of G protein-coupled receptors may be necessary for functions ranging from agonist binding, folding, maturation, stability, and internalization. Human melanocortin 2 receptor (MC2R) possesses putative N-glycosylation sites in its N-terminal extracellular domain; however, to date, the role of MC2R N-glycosylation has yet to be investigated. The objective of the present study is to examine whether N-glycosylation is essential or not for cell surface expression and cAMP production in native and MC2R accessory protein (MRAP alpha, -beta, or -dCT)-expressing cells using 293/FRT transfected with Myc-MC2R. Western blot analyses performed with or without endoglycosidase H, peptide:N-glycosidase F or tunicamycin treatments and site-directed mutagenesis revealed that MC2R was glycosylated in the N-terminal domain at its two putative N-glycosylation sites (Asn(12)-Asn(13)-Thr(14) and Asn(17)-Asn(18)-Ser(19)). In the absence of human MRAP coexpression, N-glycosylation of at least one of the two sites was necessary for MC2R cell surface expression. However, when MRAP was present, cell surface expression of MC2R mutants was either rescued entirely with the N17-18Q (QQNN) and N12-13Q (NNQQ) mutants or partially with the unglycosylated N12-13, 17-18Q (QQQQ) mutant. Functional and expression analyses revealed a discrepancy between wild-type (WT) and QQQQ cell surface receptor levels and maximal cAMP production with a 4-fold increase in EC(50) values. Taken together, these results indicate that the absence of MC2R N-glycosylation abrogates to a large extent MC2R cell surface expression in the absence of MRAPs, whereas when MC2R is N-glycosylated, it can be expressed at the plasma membrane without MRAP assistance.Endocrinology 12/2009; 151(2):660-70. · 4.72 Impact Factor