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Article Dans Une Revue Physiologia Plantarum Année : 1999

Molecular determinants of the Arabidopsis AKT1 K+ channel ionic selectivity investigated by expression in yeast of randomly mutated channels

Résumé

The Avabidopsis thaliana K+ channel AKT1 was expressed in a yeast strain defective for K+ uptake at low K+ concentrations (<3 mM). Besides restoring K+ transport in this strain, AKT1 expression increased its tolerance to salt (NaCl or LiCl), whatever the external K+ concentration used (50 mu M, 5 mM, or 50 mM), We took advantage of the latter phenomenon for screening a library of channels randomly mutated in the region that shares homologies with the pore forming domain (the so-called P domain) of animal K+ channels (Shaker family). Cassette mutagenesis was performed using a degenerate oligonucleotide that was designed to ensure, theoretically, a single mutation per P cassette. The mean number of amino acid exchanges per cassette turned out to be 1.4, Mutant channels that conferred on the transformed cells a reduction in salt tolerance (increased Na+ content, decreased K+ content, and lower growth rate, as compared to control cells expressing the wild-type channel) were selected. By co-expressing them with the wild-type AKT1 cDNA, it was shown that the mutated polypeptides were expressed, stable and correctly targeted to the cell membrane where they formed channels with altered properties. Analysis of the mutation distribution in these channels suggests that the AKT1 P domain has a structure similar to that of animal Shaker channels (a strongly constrained central region lining the tunnel that includes the highly conserved consensus motif TXXTXGYGD, and flanking regions forming the outer mouth of the pare), with an additional selectivity filter located upstream from the tunnel and formed by residues present in the N-terminal flanking region.

Dates et versions

hal-02694099 , version 1 (01-06-2020)

Identifiants

Citer

R. Ros, G. Lemaillet, A.G. Fonrouge, P. Daram, M. Enjuto, et al.. Molecular determinants of the Arabidopsis AKT1 K+ channel ionic selectivity investigated by expression in yeast of randomly mutated channels. Physiologia Plantarum, 1999, 105 (3), pp.459-468. ⟨10.1034/j.1399-3054.1999.105310.x⟩. ⟨hal-02694099⟩
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