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

The Avabidopsis thaliana K+ channel AKT1 was expressed in a yeast strain de fective 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 lib rary of channels randomly mutated in the region that shares homologies with the pore forming domain (the so-called P domain) of animal K+ channels (Sh aker family). Cassette mutagenesis was performed using a degenerate oligonu cleotide 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 reduc tion in salt tolerance (increased Na+ content, decreased K+ content, and lo wer growth rate, as compared to control cells expressing the wild-type chan nel) were selected. By co-expressing them with the wild-type AKT1 cDNA, it was shown that the mutated polypeptides were expressed, stable and correctl y targeted to the cell membrane where they formed channels with altered pro perties. Analysis of the mutation distribution in these channels suggests t hat the AKT1 P domain has a structure similar to that of animal Shaker chan nels (a strongly constrained central region lining the tunnel that includes the highly conserved consensus motif TXXTXGYGD, and flanking regions formi ng the outer mouth of the pare), with an additional selectivity filter loca ted upstream from the tunnel and formed by residues present in the N-termin al flanking region.