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

Abstract

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

The inward-rectifying K+ channel SsAKT1 is a candidate involved in K+ uptake in the halophyte Suaeda salsa under saline condition

“…5), probably because SsAKT1 conferred a higher K + uptake capacity in the yeast cells. A similar result was reported by Ros et al (1999), who showed that expressing AtAKT1 in a yeast strain 10A (trk1 − , ura3 − ) defective in high-affinity K + uptake system enhanced salt tolerance. Thus, we speculate that the up-regulation of SsAKT1 expression under saline conditions contributed to mediating significant K + uptake in roots from the external medium, providing S. salsa with the ability to maintain K + homeostasis in the plant under salinity, and ultimately contribute to its salt tolerance.…”

“…2), which are mainly expressed in roots and involved in K + uptake (Pilot et al 2003). Moreover, the yeast complementation experiments further showed that similar to AtAKT1 in Arabidopsis (Ros et al 1999;Sentenac et al 1992), SsAKT1 could mediate K + uptake over a wide range of external K + concentrations (Figs. 3 and 4); and more interestingly, expression of SsAKT1 could also enhance salt tolerance of a Na + -extruding ATPase-deficient yeast strain G19 via endowing the yeast cells with K + uptake capacity (Fig.…”

Potassium channels in plant cells

“…The broadest approaches in this context were launched for the Arabidopsis K + channels KAT1 and AKT1. For both, libraries of channels with randomly mutated selectivity filters were generated and screened in the yeast Saccharomyces cerevisiae [60–62]. Lately, a KAT1 mutant library was re‐inspected and over 200 point mutants in the pore region were examined in S. cerevisiae and Xenopus oocytes to assess the effect of the mutations on ion selectivity.…”

Sodium transport and HKT transporters: the rice model

“…In addition to the entrance through specific transporters, Na + may be taken up ectopically through transporters for K + or other ions when Na + is present at a high concentration. Assuming that typical ion concentrations prevailing in a saline soil, in which productivity is decreased, may be around 1 m m K + , 50 m m Na + , and 2 m m Ca 2+ , the putative transporter cannot be any of the well‐established root K + transporters HAK1 or AKT1 because they are not sufficiently permeable to Na + (Ros et al ., 1999; Santa‐María et al ., 1997). The transporter might be LCT1 (Amtmann et al ., 2001), or any of a collection of channels of variable K + /Na + discrimination (Amtmann and Sanders, 1999; Davenport and Tester, 2000; Demidchik et al ., 2002; Leng et al ., 2002; Maathuis and Sanders, 2001; Tyerman and Skerrett, 1999).…”