Glycosylation Affects the Protein Stability and Cell Surface Expression of Kv1.4 but Not Kv1.1 Potassium Channels

Watanabe, Itaru; Zhu, Jing; Recio-Pinto, Esperanza; Thornhill, William B.

doi:10.1074/jbc.m309802200

Cited by 100 publications

(85 citation statements)

References 16 publications

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“…It has been proposed that N-glycosylation may affect the channel stability and promote trafficking of voltagegated potassium channels to the surface (Watanabe et al, 2004). In K V 7.1 for example, a robust increase of current amplitudes seems to be due to N-glycosylation of an asparagine in the S5-H loop (Schenzer et al, 2005).…”

Section: Discussionmentioning

confidence: 99%

Temperature and pharmacological rescue of a folding-defective, dominantl-negative KV7.2 mutation associated with neonatal seizures

et al. 2011

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Benign familial neonatal seizures (BFNS) is a dominant epilepsy syndrome caused by mutations in the voltage-gated potassium channels K V 7.2 and K V 7.3. We examined the molecular pathomechanism of a BFNS-causing mutation (p.N258S) in the extracellular S5-H5 loop of K V 7.2. Wild type (WT) and mutant channels, expressed in both Xenopus laevis oocytes and CHO cells, were studied using electrophysiological techniques. The results revealed a pronounced loss-offunction with a dominant-negative effect of the mutant on WT K V 7.2 and K V 7.3 channels. Since single channel recordings of K V 7.3-K V 7.2 and K V 7.3-N285S concatemers showed similar properties for both constructs, we hypothesized that the observed reduction in current amplitude was due to a folding and trafficking defect, which was confirmed by biochemical and immunocytochemical experiments revealing a reduced number of mutant channels in the surface membrane. Furthermore, rescuing experiments revealed that upon specific incubation of transfected CHO cells -either at lower temperatures of <30 o C or in presence of the agonist retigabine -the N258S-derived currents increased 5-fold in contrast to the WT. The obtained results represent a first example of temperature and pharmacological rescue of a K V 7 mutation and suggest a folding and trafficking deficiency as the cause of reduced current amplitudes with a dominant-negative effect of N258S mutant proteins.

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Section: Discussionmentioning

confidence: 99%

Temperature and pharmacological rescue of a folding-defective, dominantl-negative KV7.2 mutation associated with neonatal seizures

et al. 2011

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“…The functional importance of attached carbohydrates on protein stability, trafficking, or function varies with different proteins. Several membrane glycoproteins such as human norepinephrine transporters (15) and voltage-gated potassium Kv1.4 channels (35) require N-linked glycosylation for correct folding. Decreased cell surface expression has been reported for hyperpolarization-activated cyclic nucleotidegated channels (19), organic anion transporter 1 (28), and dopamine transporters (12), although the role of carbohydrates in protein stability was not investigated in these studies.…”

Section: Discussionmentioning

confidence: 99%

TwoN-linked glycans are required to maintain the transport activity of the bile salt export pump (ABCB11) in MDCK II cells

Mochizuki¹,

Kagawa²,

Numari³

et al. 2007

American Journal of Physiology-Gastrointestinal and Liver Physi

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The aim of this study was to determine the role of N-linked glycosylation in protein stability, intracellular trafficking, and bile acid transport activity of the bile salt export pump [Bsep (ATP-binding cassette B11)]. Rat Bsep was fused with yellow fluorescent protein, and the following mutants, in which Asn residues of putative glycosylation sites (Asn109, Asn116, Asn122, and Asn125) were sequentially replaced with Gln, were constructed by site-directed mutagenesis: single N109Q, double N109Q + N116Q, triple N109Q + N116Q + N122Q, and quadruple N109Q + N116Q + N122Q + N125Q. Immunoblot and glycosidase cleavage analysis demonstrated that each site was glycosylated. Removal of glycans decreased taurocholate transport activity as determined in polarized MDCK II cells. This decrease resulted from rapid decay of the mutant Bsep protein; biochemical half-lives were 3.76, 3.65, 3.24, 1.35, and 0.52 h in wild-type, single-mutant, double-mutant, triple-mutant, and quadruple-mutant cells, respectively. Wild-type and single- and double-mutant proteins were distributed exclusively along the apical membranes, whereas triple- and quadruple-mutant proteins remained intracellular. MG-132 but not bafilomycin A1 extended the half-life, suggesting a role for the proteasome in Bsep degradation. To determine whether a specific glycosylation site or the number of glycans was critical for protein stability, we studied the protein expression of combinations of N-glycan-deficient mutants and observed that Bsep with one glycan was considerably unstable compared with Bsep harboring two or more glycans. In conclusion, at least two N-linked glycans are required for Bsep protein stability, intracellular trafficking, and function in the apical membrane.

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“…A VXXSL signal promotes efficient exit of Kv1.4, and a similar but less effective VXXSN sequence is operative in Kv1.5 (236,478). The cyclic nucleotide-binding domains of HERG, ERG3, and HCN2 may also act as forward-trafficking signals (9), and the Kv1.4 pore appears to harbor a pore-based forward trafficking signal (447).…”

Section: Active Export Controlmentioning

confidence: 99%

Dynamic of Ion Channel Expression at the Plasma Membrane of Cardiomyocytes

Balse

Steele

Abriel

et al. 2012

Physiological Reviews

109

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Cardiac myocytes are characterized by distinct structural and functional entities involved in the generation and transmission of the action potential and the excitation-contraction coupling process. Key to their function is the specific organization of ion channels and transporters to and within distinct membrane domains, which supports the anisotropic propagation of the depolarization wave. This review addresses the current knowledge on the molecular actors regulating the distinct trafficking and targeting mechanisms of ion channels in the highly polarized cardiac myocyte. In addition to ubiquitous mechanisms shared by other excitable cells, cardiac myocytes show unique specialization, illustrated by the molecular organization of myocyte-myocyte contacts, e.g., the intercalated disc and the gap junction. Many factors contribute to the specialization of the cardiac sarcolemma and the functional expression of cardiac ion channels, including various anchoring proteins, motors, small GTPases, membrane lipids, and cholesterol. The discovery of genetic defects in some of these actors, leading to complex cardiac disorders, emphasizes the importance of trafficking and targeting of ion channels to cardiac function. A major challenge in the field is to understand how these and other actors work together in intact myocytes to fine-tune ion channel expression and control cardiac excitability.

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Glycosylation Affects the Protein Stability and Cell Surface Expression of Kv1.4 but Not Kv1.1 Potassium Channels

Cited by 100 publications

References 16 publications

Temperature and pharmacological rescue of a folding-defective, dominantl-negative KV7.2 mutation associated with neonatal seizures

Temperature and pharmacological rescue of a folding-defective, dominantl-negative KV7.2 mutation associated with neonatal seizures

TwoN-linked glycans are required to maintain the transport activity of the bile salt export pump (ABCB11) in MDCK II cells

Dynamic of Ion Channel Expression at the Plasma Membrane of Cardiomyocytes

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