Contribution of N- and C-terminal Kv4.2 channel domains to KChIP interaction [corrected].
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.
Callsen
Britta
B
Institut für Neurale Signalverarbeitung, Zentrum für Molekulare Neurobiologie Hamburg, Germany.
Isbrandt
Dirk
D
Sauter
Kathrin
K
Hartmann
L Sven
LS
Pongs
Olaf
O
Bähring
Robert
R
D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004252:DNA Mutational Analysis; D005260:Female; D006801:Humans; D015640:Ion Channel Gating; D051605:Kv Channel-Interacting Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D008564:Membrane Potentials; D016297:Mutagenesis, Site-Directed; D018408:Patch-Clamp Techniques; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D051672:Shal Potassium Channels / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
C496752:KCNIP2 protein, human; D051605:Kv Channel-Interacting Proteins; D051672:Shal Potassium Channels