Abstract
The N-glycan pool contains high amounts of sialic acid with atypical linkage in mammalian brain. Sialoglycoconjugates are more alike in similar tissues from different mammals than in dissimilar organs from the same mammal. The intent of this study was to generate a cell model for examining the role of neuronal derived N-glycans of a voltage-gated K+ channel, Kv3.1. Neuroblastoma B35 cells were utilized to heterologously express glycosylated (wild type Kv3.1) and unglycosylated (N220Q/N229Q) forms of Kv3.1 channels. Immunoband shift assays of partially purified wild type Kv3.1 protein digested with PNgase F indicated that both sites were utilized. Additionally, the attachment of N-linked sialooligosaccharides to the wild type Kv3.1 protein was shown by digestions with neuraminidase. Endoglycosidase N digestions demonstrated that an oligo/polysialyl unit with internal α2,8-linked sialyl residues was associated with the Kv3.1 glycoprotein. To date this unusual glycosidic bond for sialyl residues has not been identified on N-glycans of potassium channels. Whole cell current measurements of glycosylated and unglycosylated Kv3.1 channels revealed differences in channel activation, inactivation and deactivation properties. Channel density at the cell surface was also greatly reduced for the unglycosylated Kv3.1 channel compared to the glycosylated Kv3.1 channel. Based on the glycosidase specificities and the immunoband patterns, our results demonstrated that both N-glycosylation sites within the S1-S2 linker of Kv3.1 are highly available, and that at least one of the carbohydrate chains is capped with an oligo/polysialyl unit. These results also provide strong evidence that the S1-S2 linker of Kv3.1 is extracytoplasmic, and that N-glycosylation modulates the inactivation and activation kinetics of the Kv3.1 channel. Given the above observations, we suggest that neuronal N-glycosylation processing of the Kv3.1 channel is crucial in regulating and fine tuning the excitable properties of neurons in the nervous system.
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