title: Distinct amino acids in the C-linker domain of the Arabidopsis K+ channel KAT2 determine its subcellular localization and activity at the plasma membrane.
abstract: Shaker K(+) channels form the major K(+) conductance of the plasma membrane in plants. They are composed of four subunits arranged around a central ion-conducting pore. The intracellular carboxy-terminal region of each subunit contains several regulatory elements, including a C-linker region and a cyclic nucleotide-binding domain (CNBD). The C-linker is the first domain present downstream of the sixth transmembrane segment and connects the CNBD to the transmembrane core. With the aim of identifying the role of the C-linker in the Shaker channel properties, we performed subdomain swapping between the C-linker of two Arabidopsis (Arabidopsis thaliana) Shaker subunits, K(+) channel in Arabidopsis thaliana2 (KAT2) and Arabidopsis thaliana K(+) rectifying channel1 (AtKC1). These two subunits contribute to K(+) transport in planta by forming heteromeric channels with other Shaker subunits. However, they display contrasting behavior when expressed in tobacco mesophyll protoplasts: KAT2 forms homotetrameric channels active at the plasma membrane, whereas AtKC1 is retained in the endoplasmic reticulum when expressed alone. The resulting chimeric/mutated constructs were analyzed for subcellular localization and functionally characterized. We identified two contiguous amino acids, valine-381 and serine-382, located in the C-linker carboxy-terminal end, which prevent KAT2 surface expression when mutated into the equivalent residues from AtKC1. Moreover, we demonstrated that the nine-amino acid stretch 312TVRAASEFA320 that composes the first C-linker α-helix located just below the pore is a crucial determinant of KAT2 channel activity. A KAT2 C-linker/CNBD three-dimensional model, based on animal HCN (for Hyperpolarization-activated, cyclic nucleotide-gated K(+)) channels as structure templates, has been built and used to discuss the role of the C-linker in plant Shaker inward channel structure and function.
authors.affiliation: Biochimie et Physiologie Moléculaire des Plantes, Institut de Biologie Intégrative des Plantes, Unité Mixte de Recherche 5004 Centre National de la Recherche Scientifique/Unité Mixte de Recherche 0386 Institut National de la Recherche Agronomique/Montpellier SupAgro/Université Montpellier 2, 34060 Montpellier cedex 2, France.
authors.forename: Manuel
authors.initials: M
authors.lastname: Nieves-Cordones
authors.forename: Alain
authors.initials: A
authors.lastname: Chavanieu
authors.forename: Linda
authors.initials: L
authors.lastname: Jeanguenin
authors.forename: Carine
authors.initials: C
authors.lastname: Alcon
authors.forename: Wojciech
authors.initials: W
authors.lastname: Szponarski
authors.forename: Sebastien
authors.initials: S
authors.lastname: Estaran
authors.forename: Isabelle
authors.initials: I
authors.lastname: Chérel
authors.forename: Sabine
authors.initials: S
authors.lastname: Zimmermann
authors.forename: Hervé
authors.initials: H
authors.lastname: Sentenac
authors.forename: Isabelle
authors.initials: I
authors.lastname: Gaillard
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000596:Amino Acids / Q000378:metabolism*; D017360:Arabidopsis / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D015640:Ion Channel Gating; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017354:Point Mutation / Q000235:genetics; D024642:Potassium Channels, Voltage-Gated / Q000737:chemistry* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D017384:Sequence Deletion / Q000235:genetics; D040681:Structural Homology, Protein; D013329:Structure-Activity Relationship; D013347:Subcellular Fractions / Q000378:metabolism
chemical_list: D000596:Amino Acids; D029681:Arabidopsis Proteins; C428126:KAT2 protein, Arabidopsis; D024642:Potassium Channels, Voltage-Gated