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Distinct Amino Acids in the C-Linker Domain of the Arabidopsis K+ Channel KAT2 Determine Its Subcellular Localization and Activity at the Plasma Membrane1[W]
- Publication Year :
- 2014
- Publisher :
- American Society of Plant Biologists, 2014.
-
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.
- Subjects :
- Models, Molecular
Arabidopsis Proteins
Amino Acid Motifs
Cell Membrane
Molecular Sequence Data
Arabidopsis
Article
Protein Structure, Tertiary
Protein Transport
Structure-Activity Relationship
Amino Acid Substitution
Potassium Channels, Voltage-Gated
Structural Homology, Protein
Point Mutation
Amino Acid Sequence
Amino Acids
Ion Channel Gating
Sequence Deletion
Subcellular Fractions
Subjects
Details
- Language :
- English
- Database :
- OpenAIRE
- Accession number :
- edsair.pmid..........fa8a1053f15ca5af8c8a9e6849885287