title: The Roles of N- and C-terminal determinants in the activation of the Kv2.1 potassium channel.
abstract: The human and rat forms of the Kv2.1 channel have identical amino acids over the membrane-spanning regions and differ only in the N- and C-terminal intracellular regions. Rat Kv2.1 activates much faster than human Kv2.1. Here we have studied the role of the N- and C-terminal residues that determine this difference in activation kinetics between the two channels. For this, we constructed mutants and chimeras between the two channels, expressed them in oocytes, and recorded currents by two-electrode voltage clamping. In the N-terminal region, mutation Q67E in the rat channel displayed a slowing of activation relative to rat wild type, whereas mutation D75E in the human channel showed faster activation than human wild type. In the C-terminal region, we found that some residues within the region of amino acids 740-853 ("CTA" domain) were also involved in determining activation kinetics. The electrophysiological data also suggested interactions between the N and C termini. Such an interaction was confirmed directly by using a glutathione S-transferase (GST) fusion protein with the N terminus of Kv2.1, which we showed to bind to the C terminus of Kv2.1. Taken together, these data suggest that exposed residues in the T1 domain of the N terminus, as well as the CTA domain in the C terminus, are important in determining channel activation kinetics and that these N- and C-terminal regions interact.
authors.affiliation: School of Biomedical Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom.
authors.forename: Min
authors.initials: M
authors.lastname: Ju
authors.forename: Louisa
authors.initials: L
authors.lastname: Stevens
authors.forename: Emma
authors.initials: E
authors.lastname: Leadbitter
authors.forename: Dennis
authors.initials: D
authors.lastname: Wray
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D003001:Cloning, Molecular; D051668:Delayed Rectifier Potassium Channels; D005260:Female; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D007700:Kinetics; D008564:Membrane Potentials / Q000502:physiology; D008565:Membrane Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins; D009865:Oocytes / Q000502:physiology; D010446:Peptide Fragments / Q000737:chemistry*; D015221:Potassium Channels / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D024642:Potassium Channels, Voltage-Gated; D011487:Protein Conformation; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D051669:Shab Potassium Channels; D014982:Xenopus laevis
chemical_list: D051668:Delayed Rectifier Potassium Channels; C496988:KCNB1 protein, human; C120777:KCNV1 protein, human; C496991:Kcnb1 protein, rat; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D011993:Recombinant Fusion Proteins; D051669:Shab Potassium Channels; D005982:Glutathione Transferase