Membrane Distribution and Activity of a Neuronal Voltage-Gated K+ Channel is Modified by Replacement of Complex Type N-Glycans with Hybrid Type

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.

Knockdown of N-Acetylglucosaminyltransferase-II Reduces Matrix Metalloproteinase 2 Activity and Suppresses Tumorigenicity in Neuroblastoma Cell Line

“…Human BE(2)-C (HuNB) and rat B35 (NB_1) neuroblastoma (NB) cells were purchased from American Type Culture Collection (Manassas, VA, USA) and used to engineer the human, HuNB(-MGAT2), and rat, NB_1(-MGAT2) glycosylation mutant cell lines, and also, NB cell lines stably expressing wild-type (WT) or N220/9Q (DM) Kv3.1b proteins [13,25]. Cultured NB cell lines were maintained in DMEM, including 10% FBS, 50 U/mL penicillin, and 50 µg/mL streptomycin at 37 • in a 5% CO 2 atmosphere.…”

“…Cultured NB cell lines were maintained in DMEM, including 10% FBS, 50 U/mL penicillin, and 50 µg/mL streptomycin at 37 • in a 5% CO 2 atmosphere. In short, CRISPR-Cas9 technology was used to silence MGAT2 in the HuNB cell line to produce HuNB(-MGAT2), as we previously described for creating the rat NB_1(-MGAT2) cell line [25]. Of note, the sgRNA oligonucleotides (5 -CACCGTTCCGCATCTACAAACGGA-3 and 5 -AAACTCCGTTTGTAGATGCGGAAC-3 ) were identical to those used to silence MGAT2 in Chinese hamster [24] and rat [25] cells.…”

“…In short, CRISPR-Cas9 technology was used to silence MGAT2 in the HuNB cell line to produce HuNB(-MGAT2), as we previously described for creating the rat NB_1(-MGAT2) cell line [25]. Of note, the sgRNA oligonucleotides (5 -CACCGTTCCGCATCTACAAACGGA-3 and 5 -AAACTCCGTTTGTAGATGCGGAAC-3 ) were identical to those used to silence MGAT2 in Chinese hamster [24] and rat [25] cells. Sequencing of the genomic DNA fragment verified that the gene was silenced from nine separate cell clones.…”

“…Total cell membrane and whole cell lysate samples were evaluated by western and lectin blotting [25,29]. Samples of HuNB(-MGAT2) cells transiently expressing MGAT2 were harvested at about 72 h for Western and lectin blots.…”

“…The CRISPR/Cas9 technique [32] was employed to engineer a human NB (HuNB) cell line with MGAT2 (encodes GnT-II) silenced, referred to as HuNB(-MGAT2). Previously, we used this technique to construct a rat NB (NB_1) cell line lacking GnT-II, called NB_1(-MGAT2) [25]. Since DNA sequences are conserved in this region between human and rat, the gRNA pair employed was identical.…”