TY  - JOUR
A1  - Dreyer, Ingo
A1  - Poree, Fabien
A1  - Schneider, A.
A1  - Mittelstadt, J.
A1  - Bertl, Adam
A1  - Sentenac, H.
A1  - Thibaud, Jean-Baptiste
A1  - Müller-Röber, Bernd
T1  - Assembly of plant Shaker-like K-out channels requires two distinct sites of the channel alpha-subunit
N2  - 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 thatchannel a-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
Y1  - 2004
SN  - 0006-3495
ER  - 
TY  - JOUR
A1  - Poree, Fabien
A1  - Wulfetange, K.
A1  - Naso, A.
A1  - Carpaneto, Armando
A1  - Roller, A.
A1  - Natura, G.
A1  - Bertl, Adam
A1  - Sentenac, H.
A1  - Thibaud, Jean-Baptiste
A1  - Dreyer, Ingo
T1  - Plant K-in and K-out channels : Approaching the trait of opposite rectification by analyzing more than 250 KAT1- SKOR chimeras
N2  - Members of the Shaker-like plant K+ channel family share a common structure, but are highly diverse in their function: they behave as either hyperpolarization-activated inward-rectifying (K-in) channels, or leak-like (K-weak) channels, or depolarization-activated outward-rectifying (K-out) channels. Here we created 256 chimeras between the K-in channel KAT1 and the K-out channel SKOR. The chimeras were screened in a potassium-uptake deficient yeast strain to identify those, which mediate potassium inward currents, i.e., which are functionally equivalent to KAT1. This strategy allowed Lis to identify three chimeras which differ from KAT1 in three parts of the polypeptide: the cytosolic N- terminus, the cytosolic C-terminus, and the putative voltage-sensor S4. Additionally, mutations in the K-out Channel SKOR were generated in order to localize molecular entities underlying its depolarization activation. The triple mutant SKOR-D312N-M313L-1314G, carrying amino-acid changes in the S6 segment, was identified as a channel which did not display any rectification in the tested voltage-range. (C) 2005 Elsevier Inc. All rights reserved
Y1  - 2005
SN  - 0006-291X
ER  -