Assembly of plant Shaker-like K(out) channels requires two distinct sites of the channel alpha-subunit.
SKOR and GORK are outward-rectifying plant potassium channels from Arabidopsis thaliana. They belong to the Shaker superfamily of voltage-dependent K(+) channels. Channels of this class are composed of four alpha-subunits and subunit assembly is a prerequisite for channel function. In this study the assembly mechanism of SKOR was investigated using the yeast two-hybrid system and functional assays in Xenopus oocytes and in yeast. We demonstrate that SKOR and GORK physically interact and assemble into heteromeric K(out) channels. Deletion mutants and chimeric proteins generated from SKOR and the K(in) channel alpha-subunit KAT1 revealed that the cytoplasmic C-terminus of SKOR determines channel assembly. Two domains that are crucial for channel assembly were identified: i), a proximal interacting region comprising a putative cyclic nucleotide-binding domain together with 33 amino acids just upstream of this domain, and ii), a distal interacting region showing some resemblance to the K(T) domain of KAT1. Both regions contributed differently to channel assembly. Whereas the proximal interacting region was found to be active on its own, the distal interacting region required an intact proximal interacting region to be active. K(out) alpha-subunits did not assemble with K(in) alpha-subunits because of the absence of interaction between their assembly sites.
Biochimie et Physiologie Moléculaires des Plantes, UMR 5004, Agro-M/CNRS/INRA/UM2, F-34060 Montpellier Cedex 1, France. dreyer@rz.uni-potsdam.de
Ingo
I
Dreyer
Fabien
F
Porée
Antje
A
Schneider
Jessica
J
Mittelstädt
Adam
A
Bertl
Hervé
H
Sentenac
Jean-Baptiste
JB
Thibaud
Bernd
B
Mueller-Roeber
D000818:Animals; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002478:Cells, Cultured; D015640:Ion Channel Gating / Q000502:physiology; D008564:Membrane Potentials / Q000502:physiology; D009865:Oocytes / Q000737:chemistry / Q000378:metabolism*; D015221:Potassium Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D051637:Shaker Superfamily of Potassium Channels; D013329:Structure-Activity Relationship; D014982:Xenopus laevis; D015003:Yeasts / Q000737:chemistry / Q000378:metabolism*
D029681:Arabidopsis Proteins; D015221:Potassium Channels; D021122:Protein Subunits; D011994:Recombinant Proteins; C114544:SKOR protein, Arabidopsis; D051637:Shaker Superfamily of Potassium Channels