Genetic selection of inward-rectifying K+ channel mutants with reduced Cs+ sensitivity by random recombinant DNA shuffling mutagenesis and mutant selection in yeast

Structure-function relationships of voltage-dependent ion channels have been analysed by site-directed mutagenesis and functional electrophysiological characterizations. A complementary genetic selection approach for identifying interesting K+ channel mutations, allowing selection from a large random pool of K+ channel mutants, is described here, Noninactivating inward-rectifying potassium channels provide an important mechanism for K+ uptake into plant cells. The Arabidopsis K-in(+) channel, KAT1, functionally complements a yeast strain deficient in K+ uptake. The alkali metal Cs+ blocks K-in(+) channels and inhibits growth of yeast cells expressing KAT1. In this study a mutagenesis method called 'DNA shuffling' (90 'recombinant PCR') was applied to generate random mutants in the KAT1 channel. Randomly mutated libraries of KAT1 were expressed in yeast and Cs+-resistant colonies were selected, KAT1 mutants that conferred a Cs+-resistant phenotype for yeast growth were functionally characterized by expression in Xenopus oocytes and two electrode voltage clamp analysis. K+ channel properties, such as Cs+-block sensitivity, cation selectivity, and steady-state activation were altered by mutating amino acids in the pore region, but also in regions adjacent to the pore region of the KAT1 channel, Amino acid substitutions previously not targeted for site-directed mutagenesis were identified that affect Cs+ block of K+ channels. Two amino acid positions, I209 in the S5 domain and E269, were mutated in more than one of the Cs+-resistant mutants indicating important roles in Cs+ sensitivity. Shifts in steady-state activation and/or resistance to block by Cs+ were determined to be mechanisms which contribute to the Cs+ resistance of the selected mutants.