Abstract
Abnormal modifications in N-glycosylation processing are commonly associated with neurological disorders, although the impact of specific N-glycans on neuronal excitability is unknown. By replacement of complex types of N-glycans with hybrid types in neuroblastoma cells, we provide the first study that addresses how distinct N-glycan types impact neuronal excitability. Using CRISPR/Cas9 technology, NB_1, a clonal cell line derived from rat neuroblastoma cells (NB), was modified to create an N-glycosylation mutant cell line, NB_1 (-Mgat2), which expresses predominantly hybrid type N-glycans. Western and lectin blotting, flow cytometry, TIRF and DIC microscopy, and patch clamp studies were conducted. Lectin binding revealed the predominant type of N-glycans expressed in NB_1 (-Mgat2) is hybrid while those of NB and NB_1 are complex. Kv3.1 b-expressing cells with complex N-glycans localized more glycosylated Kv3.1b to the neurites than cells with hybrid N-glycans. Further the absence of N-glycan attachment to Kv3.1b was critical for sub-plasma distribution of Kv3.1b to neurites in primary adult mammalian neurons, along with NB cells. Replacement of complex type N-glycans with hybrid type hindered the opening and closing rates of outward ionic currents of Kv3.1 b-expressing NB cells. The lacks of N-glycan attachment hindered the rates even more but were not significantly different between the NB cell lines. Taken together, our evidence supports N-glycosylation impacts the sub-plasma membrane localization and activity of Kv3.1 b-containing channels. We propose that N-glycosylation processing of Kv3.1 b-containing channels contributes to neuronal excitability, and abnormal modifications in N-glycosylation processing of Kv3.1b could contribute to neurological diseases.
Highlights
N-glycans are a part of glycoproteins and are classified as three major types: oligomannose, hybrid, and complex, each of which is composed of a common core sugar sequence [1]
The role of N-glycosylation processing of Kv3.1 in modifying neuronal excitability was examined by replacement of complex N-glycans with hybrid type N-glycans in rat neuroblastoma (NB) cells
A recent study reports that N-glycans of Kv3.1b regulate cell surface expression since the cell surface expression is reduced 10-fold for the unglycosylated Kv3.1b protein compared to its glycosylated counterpart [44]
Summary
N-glycans are a part of glycoproteins and are classified as three major types: oligomannose, hybrid, and complex, each of which is composed of a common core sugar sequence [1]. Nacetylglucosaminyltransferases (GlcNAcTs, coded by Mgat genes) act to initiate the different branch points onto the common core, thereby creating the three major types of glycans [1]. Nglycosylation is vital to the function of cells, and aberrant N-glycosylation processing can lead to numerous pathophysiological conditions. The relevance of N-glycosylation site occupancy of both Kv3.1b and 3.1a proteins in cell function has been highlighted in studies that demonstrate N-glycans contribute to the opening and closing of Kv3 channels with Kv3.1b, as well as the spatial localization of Kv3.1b and 3.1a proteins in the plasma membrane, of Sf9 insect [15], CHO [16], and B35 neuroblastoma cells [17,18,19]. It is likely that unoccupied N-glycosylation sites of Kv channels would perturb neural excitability
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