Site-directed Glycosylation Tagging of Functional Kir2.1 Reveals That the Putative Pore-forming Segment Is Extracellular

Schwalbe, Ruth A.; Rudin, Alicia; Xia, Shen‐Ling; Wingo, Charles S.

doi:10.1074/jbc.m201668200

Cited by 12 publications

(18 citation statements)

References 32 publications

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“…3B), a major band of ϳ50 kDa was observed without microsomes, the expected size for Kir2.1. With microsomes present there did not appear to be a major increase in size, suggesting low or no posttranslational modification by glycosylation in agreement with reports that Kir2.1 is not glycosylated (53). The Kir2.1 protein observed on Western blots would therefore seem to be the unglycosylated form.…”

Section: Resultssupporting

confidence: 82%

Gastric parietal cell secretory membrane contains PKA- and acid-activated Kir2.1 K⁺ channels

Malinowska

Sherry

Tewari

et al. 2004

American Journal of Physiology-Cell Physiology

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Our objective was to identify and localize a K+ channel involved in gastric HCl secretion at the parietal cell secretory membrane and to characterize and compare the functional properties of native and recombinant gastric K+ channels. RT-PCR showed that mRNA for Kir2.1 was abundant in rabbit gastric mucosa with lesser amounts of Kir4.1 and Kir7.1, relative to beta-actin. Kir2.1 mRNA was localized to parietal cells of rabbit gastric glands by in situ RT-PCR. Resting and stimulated gastric vesicles contained Kir2.1 by Western blot analysis at approximately 50 kDa as observed with in vitro translation. Immunoconfocal microscopy showed that Kir2.1 was present in parietal cells, where it colocalized with H+ -K+ -ATPase and ClC-2 Cl- channels. Function of native K+ channels in rabbit resting and stimulated gastric mucosal vesicles was studied by reconstitution into planar lipid bilayers. Native gastric K+ channels exhibited a linear current-voltage relationship and a single-channel slope conductance of approximately 11 pS in 400 mM K2SO4. Channel open probability (Po) in stimulated vesicles was high, and that of resting vesicles was low. Reduction of extracellular pH plus PKA treatment increased resting channel Po to approximately 0.5 as measured in stimulated vesicles. Full-length rabbit Kir2.1 was cloned. When stably expressed in Chinese hamster ovary (CHO) cells, it was activated by reduced extracellular pH and forskolin/IBMX with no effects observed in nontransfected CHO cells. Cation selectivity was K+ = Rb+ >> Na+ = Cs+ = Li+ = NMDG+. These findings strongly suggest that the Kir2.1 K+ channel may be involved in regulated gastric acid secretion at the parietal cell secretory membrane.

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Section: Resultssupporting

confidence: 82%

Gastric parietal cell secretory membrane contains PKA- and acid-activated Kir2.1 K⁺ channels

Malinowska

Sherry

Tewari

et al. 2004

American Journal of Physiology-Cell Physiology

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“…Sf9 cells were maintained in Hink's TNM‐FH medium containing 10% FBS, 10 µg·mL −1 gentamicin, and 0.1% Pluronic F‐68 at 27 °C as previously described [50]. Monolayer Sf9 cultures were used to maintain Sf9 cells and were passaged about twice a week.…”

Section: Methodsmentioning

confidence: 99%

Characterization of N‐glycosylation consensus sequences in the Kv3.1 channel

2006

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N‐Glycosylation is a cotranslational and post‐translational process of proteins that may influence protein folding, maturation, stability, trafficking, and consequently cell surface expression of functional channels. Here we have characterized two consensus N‐glycosylation sequences of a voltage‐gated K+ channel (Kv3.1). Glycosylation of Kv3.1 protein from rat brain and infected Sf9 cells was demonstrated by an electrophoretic mobility shift assay. Digestion of total brain membranes with peptide N glycosidase F (PNGase F) produced a much faster‐migrating Kv3.1 immunoband than that of undigested brain membranes. To demonstrate N‐glycosylation of wild‐type Kv3.1 in Sf9 cells, cells were treated with tunicamycin. Also, partially purified proteins were digested with either PNGase F or endoglycosidase H. Attachment of simple‐type oligosaccharides at positions 220 and 229 was directly shown by single (N229Q and N220Q) and double (N220Q/N229Q) Kv3.1 mutants. Functional measurements and membrane fractionation of infected Sf9 cells showed that unglycosylated Kv3.1s were transported to the plasma membrane. Unitary conductance of N220Q/N229Q was similar to that of the wild‐type Kv3.1. However, whole cell currents of N220Q/N229Q channels had slower activation rates, and a slight positive shift in voltage dependence compared to wild‐type Kv3.1. The voltage dependence of channel activation for N229Q and N220Q was much like that for N220Q/N229Q. These results demonstrate that the S1–S2 linker is topologically extracellular, and that N‐glycosylation influences the opening of the voltage‐dependent gate of Kv3.1. We suggest that occupancy of the sites is critical for folding and maturation of the functional Kv3.1 at the cell surface.

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“…Acetyl-D-mannosamine, N-[6-3 H] is a precursor of sialic acid and is commonly used to radiolabel sialylated N-glycans [28]. h, the unradiolabelled and radiolabelled transfected cells were harvested and Kv3.1 was immunoaffinity-purified in a similar manner to that previously described by us [21,29]. In short, transfected B35 cells were resuspended in lysis buffer (50 mM Na 2 HPO 4 , 0.3 M KCl, pH 7.5 …”

Section: Transient Tranfections Of B35 Cells and Metabolic Radiolabelmentioning

confidence: 99%

Atypical sialylated N-glycan structures are attached to neuronal voltage-gated potassium channels

Cartwright

Schwalbe

2009

Bioscience Reports

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Mammalian brains contain relatively high amounts of common and uncommon sialylated N-glycan structures. Sialic acid linkages were identified for voltage-gated potassium channels, Kv3.1, 3.3, 3.4, 1.1, 1.2 and 1.4, by evaluating their electrophoretic migration patterns in adult rat brain membranes digested with various glycosidases. Additionally, their electrophoretic migration patterns were compared with those of NCAM (neural cell adhesion molecule), transferrin and the Kv3.1 protein heterologously expressed in B35 neuroblastoma cells. Metabolic labelling of the carbohydrates combined with glycosidase digestion reactions were utilized to show that the N-glycan of recombinant Kv3.1 protein was capped with an oligo/poly-sialyl unit. All three brain Kv3 glycoproteins, like NCAM, were terminated with alpha2,3-linked sialyl residues, as well as atypical alpha2,8-linked sialyl residues. Additionally, at least one of their antennae was terminated with an oligo/poly-sialyl unit, similar to recombinant Kv3.1 and NCAM. In contrast, brain Kv1 glycoproteins consisted of sialyl residues with alpha2,8-linkage, as well as sialyl residues linked to internal carbohydrate residues of the carbohydrate chains of the N-glycans. This type of linkage was also supported for Kv3 glycoproteins. To date, such a sialyl linkage has only been identified in gangliosides, not N-linked glycoproteins. We conclude that all six Kv channels (voltage-gated K+ channels) contribute to the alpha2,8-linked sialylated N-glycan pool in mammalian brain and furthermore that their N-glycan structures contain branched sialyl residues. Identification of these novel and unique sialylated N-glycan structures implicate a connection between potassium channel activity and atypical sialylated N-glycans in modulating and fine-tuning the excitable properties of neurons in the nervous system.

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Site-directed Glycosylation Tagging of Functional Kir2.1 Reveals That the Putative Pore-forming Segment Is Extracellular

Cited by 12 publications

References 32 publications

Gastric parietal cell secretory membrane contains PKA- and acid-activated Kir2.1 K⁺ channels

Gastric parietal cell secretory membrane contains PKA- and acid-activated Kir2.1 K⁺ channels

Characterization of N‐glycosylation consensus sequences in the Kv3.1 channel

Atypical sialylated N-glycan structures are attached to neuronal voltage-gated potassium channels

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Site-directed Glycosylation Tagging of Functional Kir2.1 Reveals That the Putative Pore-forming Segment Is Extracellular

Cited by 12 publications

References 32 publications

Gastric parietal cell secretory membrane contains PKA- and acid-activated Kir2.1 K+ channels

Gastric parietal cell secretory membrane contains PKA- and acid-activated Kir2.1 K+ channels

Characterization of N‐glycosylation consensus sequences in the Kv3.1 channel

Atypical sialylated N-glycan structures are attached to neuronal voltage-gated potassium channels

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Gastric parietal cell secretory membrane contains PKA- and acid-activated Kir2.1 K⁺ channels

Gastric parietal cell secretory membrane contains PKA- and acid-activated Kir2.1 K⁺ channels