Kvβ Subunits Increase Expression of Kv4.3 Channels by Interacting with Their C Termini

Yang, Eun Kyoung; Alvira, Mauricio R.; Levitan, Edwin S.; Takimoto, Koichi

doi:10.1074/jbc.m004768200

Cited by 85 publications

(77 citation statements)

References 29 publications

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“…The yield of correctly folded truncated Shaker channels after affinity purification was 42% of the total protein, compared with Ϸ30% for the WT channel (17). Coexpression of either full-length or truncated channel with rat Kv␤2 increased surface expression by Ϸ25%, consistent with immunofluorescent staining data (21,38). Binding of Kv␤ does not change the comparative yields of detergent purified channels (Table 1).…”

Section: Resultssupporting

confidence: 67%

Conformational changes in the C terminus of Shaker K ⁺ channel bound to the rat Kvβ2-subunit

Sokolova

Accardi

Gutiérrez

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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We studied the structure of the C terminus of the Shaker potassium channel. The 3D structures of the full-length and a C-terminal deletion (⌬C) mutant of Shaker were determined by electron microscopy and single-particle analysis. The difference map between the full-length and the truncated channels clearly shows a compact density, located on the sides of the T1 domain, that corresponds to a large part of the C terminus. We also expressed and purified both WT and ⌬C Shaker, assembled with the rat Kv␤2-subunit. By using a difference map between the full-length and truncated Shaker ␣-␤ complexes, a conformational change was identified that shifts a large part of the C terminus away from the membrane domain and into close contact with the ␤-subunit. This conformational change, induced by the binding of the Kv␤2-subunit, suggests a possible mechanism for the modulation of the K ؉ voltage-gated channel function by its ␤-subunit.C-terminal deletion ͉ electron microscopy ͉ single-particle analysis

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Section: Resultssupporting

confidence: 67%

Conformational changes in the C terminus of Shaker K ⁺ channel bound to the rat Kvβ2-subunit

Sokolova

Accardi

Gutiérrez

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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“…Immunoblot analysis was performed following SDS-PAGE with polyclonal anti-Myc, anti-GFP (MBL International), or anti-KChIP2 antibodies as described previously (12). Anti-KChIP2 antibody was generated against a synthetic peptide containing the first 25 amino acids of the protein and was affinity-purified.…”

Section: Methodsmentioning

confidence: 99%

“…3 H]palmitic acid in the standard medium supplemented with extra sodium pyruvate (200 mg/ liter) for 4 h. Cell extracts were prepared, and immunoprecipitation was performed with anti-GFP antibody, as described previously (12,27), except that the solutions for binding and washing were done with the modified RIPA buffer. Precipitated materials were separated on a 10% SDS gel and subjected to fluorographic exposure.…”

Section: Methodsmentioning

confidence: 99%

“…The expression and localization of Kv4 channels can be regulated by various channel-interacting proteins including Kv␤ subunits (11,12), MiRP1 (13), the chaperon-like proteins KChAPs (14), and the cytoskeleton-associated proteins filamins (15). More recently, a class of EF hand-containing Ca 2ϩ -binding proteins (K ϩ channel-interacting proteins (KChIPs)) 1 has been identified as physiologically important auxiliary subunits for Kv4 channels (16).…”

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confidence: 99%

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Palmitoylation of KChIP Splicing Variants Is Required for Efficient Cell Surface Expression of Kv4.3 Channels

Takimoto¹,

Yang²,

Conforti³

2002

Journal of Biological Chemistry

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129

121

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The Ca 2؉ -binding proteins KChIP1-4 (KChIP3 is also known as DREAM and calsenilin) act as auxiliary subunits for voltage-gated K ؉ channels in the Kv4 family. Here we identify three splicing isoforms of rat KChIP2 with variable N-terminal peptides. The two longer isoforms, which contain the 32-amino acid peptide, produce larger increases in Kv4.3 protein level and current density and more effectively localize themselves and their associated channels at the plasma membrane than the shortest variant. The 32-amino acid peptide contains potential palmitoylation cysteines. Metabolic labeling demonstrates that these cysteines in the KChIP2 isoforms, as well as the corresponding sites in KChIP3, are palmitoylated. Mutating these cysteines reduces their plasma membrane localization and the enhancement of Kv4.3 current density. Thus, palmitoylation of the KChIP auxiliary subunits controls plasma membrane localization of their associated channels.

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“…Constructions-Rat KChIP2 splicing variant (GenBank TM accession numbers AF269283, 269284, and 269285) and NCS-1 cDNAs were subcloned into an Emerald-C1 expression vector that had been constructed by replacing the enhanced green fluorescent protein of EGFP-C1 (Clontech) with Emerald (Packard) by a PCR-based method, as described previously (8,13). Myc-tagged Kv1.4, Kv2.1, and Kv4.3 cDNAs were generated by subcloning full-length channel cDNAs into pCS2ϩMT.…”

Section: Methodsmentioning

confidence: 99%

Effective Association of Kv Channel-interacting Proteins with Kv4 Channel Is Mediated with Their Unique Core Peptide

Ren

Shand

Takimoto

2003

Journal of Biological Chemistry

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Kv channel-interacting proteins (KChIPs) and neuronal calcium sensor-1 (NCS-1) have been shown to interact with Kv4 channel ␣-subunits to regulate the expression and/or gating of these channels. Here we examine the specificity and sites of these proteins for interaction with Kv channel proteins. Immunoprecipitation and green fluorescent protein imaging show that KChIPs (but not NCS-1) effectively bind to Kv4.3 protein and localize at the plasma membrane when channel proteins are coexpressed. Analysis with chimeric proteins between KChIP2 and NCS-1 reveals that the three regions of KChIP2 (the linker between the first and second EF hands, the one between the third and fourth EF hands, and the C-terminal peptide after the fourth EF hand) are necessary and sufficient for its effective binding to Kv4.3 protein. The chimera with these three KChIP2 portions slowed inactivation and facilitated recovery from inactivation of Kv4.3 current. These results indicate that the sequence difference in these three regions between KChIPs and NCS-1 determines the specificity and affinity for interaction with Kv4 protein. Because the three identified regions surround the large hydrophobic crevice based on the NCS-1 crystal structure, this crevice may be the association site of KChIPs for the channel protein.

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Kvβ Subunits Increase Expression of Kv4.3 Channels by Interacting with Their C Termini

Cited by 85 publications

References 29 publications

Conformational changes in the C terminus of Shaker K ⁺ channel bound to the rat Kvβ2-subunit

Conformational changes in the C terminus of Shaker K ⁺ channel bound to the rat Kvβ2-subunit

Palmitoylation of KChIP Splicing Variants Is Required for Efficient Cell Surface Expression of Kv4.3 Channels

Effective Association of Kv Channel-interacting Proteins with Kv4 Channel Is Mediated with Their Unique Core Peptide

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Kvβ Subunits Increase Expression of Kv4.3 Channels by Interacting with Their C Termini

Cited by 85 publications

References 29 publications

Conformational changes in the C terminus of Shaker K + channel bound to the rat Kvβ2-subunit

Conformational changes in the C terminus of Shaker K + channel bound to the rat Kvβ2-subunit

Palmitoylation of KChIP Splicing Variants Is Required for Efficient Cell Surface Expression of Kv4.3 Channels

Effective Association of Kv Channel-interacting Proteins with Kv4 Channel Is Mediated with Their Unique Core Peptide

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Conformational changes in the C terminus of Shaker K ⁺ channel bound to the rat Kvβ2-subunit

Conformational changes in the C terminus of Shaker K ⁺ channel bound to the rat Kvβ2-subunit