Figure 2

Mutating the pore domain of the Arabidopsis potassium channel AKT1 and functional consequences in vivo . (A) Wild type AKT1 in the Gateway® Entry vector pDONR207 is used as template for SDM of the GYG-motif within the pore-domain of the voltage-gated potassium channel AKT1 which is critical for formation of the channel-pore. SDM-Assist suggested primers for a single mutation of the Glycine 255 to Threonine adding a SnaBI-site, and for mutating both Glycines to Alanine introducing an NdeI site within the gene. Both restriction sites were absent in the wild type sequence. (B) DNA restriction analysis of randomly picked colonies after SDM (for PCR conditions, see Material and methods) demonstrates that distinction between wild type (lane 1 and 4) and mutated sequences (lane 2 and 3) becomes easily possible through the addition of a restriction site: an additional band appears in mutated plasmids (arrows). Sequence verification (trace file) is incorporated in (A). (C) Functional consequence of the single or double mutation: The yeast strain SGY1528 [22] which is disrupted in potassium uptake can only grow on high K⁺-medium (+100 mM) or on low K⁺-medium (0.5 mM) if an exogenous K⁺ transporter (such as wild type AKT1) is expressed. Disruption of the GYG-motif in the mutant AKT1-G255T and G255A;G257A renders the channel dysfunctional on low K⁺-medium, the yeast fails to grow. Equal amounts of yeast were dropped as dilution series on CSM-Leu^-, His^-, Trp^- media with final potassium concentrations of 100 mM or 0.5 mM. Growth on plates with high K⁺-concentration was monitored after 2 days and on low K⁺-plates after 8 days at 30°C. AKT1 and mutants were expressed using the yeast vector pMetYC-Dest [21].