title: The subfamily-specific interaction between Kv2.1 and Kv6.4 subunits is determined by interactions between the N- and C-termini.
abstract: The "silent" voltage-gated potassium (KvS) channel subunit Kv6.4 does not form electrically functional homotetramers at the plasma membrane but assembles with Kv2.1 subunits, generating functional Kv2.1/Kv6.4 heterotetramers. The N-terminal T1 domain determines the subfamily-specific assembly of Kv1-4 subunits by preventing interactions between subunits that belong to different subfamilies. For Kv6.4, yeast-two-hybrid experiments showed an interaction of the Kv6.4 N-terminus with the Kv2.1 N-terminus, but unexpectedly also with the Kv3.1 N-terminus. We confirmed this interaction by Fluorescence Resonance Energy Transfer (FRET) and co-immunoprecipitation (co-IP) using N-terminal Kv3.1 and Kv6.4 fragments. However, full-length Kv3.1 and Kv6.4 subunits do not form heterotetramers at the plasma membrane. Therefore, additional interactions between the Kv6.4 and Kv2.1 subunits should be important in the Kv2.1/Kv6.4 subfamily-specificity. Using FRET and co-IP approaches with N- and C-terminal fragments we observed that the Kv6.4 C-terminus physically interacts with the Kv2.1 N-terminus but not with the Kv3.1 N-terminus. The N-terminal amino acid sequence CDD which is conserved between Kv2 and KvS subunits appeared to be a key determinant since charge reversals with arginine substitutions abolished the interaction between the N-terminus of Kv2.1 and the C-terminus of both Kv2.1 and Kv6.4. In addition, the Kv6.4(CKv3.1) chimera in which the C-terminus of Kv6.4 was replaced by the corresponding domain of Kv3.1, disrupted the assembly with Kv2.1. These results indicate that the subfamily-specific Kv2.1/Kv6.4 heterotetramerization is determined by interactions between Kv2.1 and Kv6.4 that involve both the N- and C-termini in which the conserved N-terminal CDD sequence plays a key role.
authors.affiliation: Laboratory for Molecular Biophysics, Physiology and Pharmacology, Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium.
authors.forename: Elke
authors.initials: E
authors.lastname: Bocksteins
authors.affiliation: Laboratory for Molecular Biophysics, Physiology and Pharmacology, Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium.
authors.forename: Evy
authors.initials: E
authors.lastname: Mayeur
authors.affiliation: Laboratory for Molecular Biophysics, Physiology and Pharmacology, Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium.
authors.forename: Abbi
authors.initials: A
authors.lastname: Van Tilborg
authors.affiliation: Laboratory for Molecular Biophysics, Physiology and Pharmacology, Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium.
authors.forename: Glenn
authors.initials: G
authors.lastname: Regnier
authors.affiliation: Laboratory of Cell Biology and Histology, Department of Veterinary Sciences, University of Antwerp, Antwerp, Belgium.
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Timmermans
authors.affiliation: Laboratory for Molecular Biophysics, Physiology and Pharmacology, Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium.
authors.forename: Dirk J
authors.initials: DJ
authors.lastname: Snyders
mesh_terms: D000595:Amino Acid Sequence; D031541:Fluorescence Resonance Energy Transfer; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation; D016296:Mutagenesis; D018408:Patch-Clamp Techniques; D011485:Protein Binding; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D051669:Shab Potassium Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D051669:Shab Potassium Channels