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Contribution of N‐ and C‐terminal channel domains to Kv channel interacting proteins in a mammalian cell line
Author(s) -
Callsen Britta,
Isbrandt Dirk,
Sauter Kathrin,
Hartmann L. Sven,
Pongs Olaf,
Bähring Robert
Publication year - 2005
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1113/jphysiol.2005.094359
Subject(s) - gating , cytoplasm , mutagenesis , gene isoform , c terminus , biophysics , mutant , binding site , potassium channel , amino acid , hek 293 cells , microbiology and biotechnology , plasma protein binding , chemistry , alanine scanning , alanine , biochemistry , biology , gene
Association of Shal gene‐related voltage‐gated potassium (Kv4) channels with cytoplasmic Kv channel interacting proteins (KChIPs) influences inactivation gating and surface expression. We investigated both functional and biochemical consequences of mutations in cytoplasmic N and C‐terminal Kv4.2 domains to characterize structural determinants for KChIP interaction. We performed a lysine‐scanning mutagenesis within the proximal 40 amino acid portion and a structure‐based mutagenesis in the tetramerization 1 (T1) domain of Kv4.2. In addition, the cytoplasmic Kv4.2 C‐terminus was truncated at various positions. Wild‐type and mutant Kv4.2 channels were coexpressed with KChIP2 isoforms in mammalian cell lines. The KChIP2‐induced modulation of Kv4.2 currents was studied with whole‐cell patch clamp and the binding of KChIP2 isoforms to Kv4.2 channels with coimmunoprecipitation experiments. Our results define one major interaction site for KChIPs, including amino acids in the proximal N‐terminus between residues 11 and 23, where binding and functional modulation are essentially equivalent. A further interaction site includes residues in the T1 domain. Notably, C‐terminal deletions also had marked effects on KChIP2‐dependent gating modulation and KChIP2 binding, revealing a previously unknown involvement of domains within the cytoplasmic Kv4.2 C‐terminus in KChIP interaction. Less coincidence of binding and functional modulation indicates a more loose ‘anchoring’ at T1‐ and C‐terminal interaction sites. Our results refine and extend previously proposed structural models for Kv4.2/KChIP complex formation.