The ability to express heterologous proteins in K+ uptake-defective strains of Saccharomyces cerevisiae can be exploited to identify cDNAs encoding heterologous K+ channels. Moreover, the ability of heterologous potassium channels like KAT1 and AKT1 to suppress completely the conditional negative growth phenotype of S. cerevisiae cells containing mutations in TRK1 or TRK1 and TRK2 opens the field of plant K+ channel biology to molecular approaches. Owing to the efficiency of modern techniques in molecular biology structure/function studies of K+ channels involving site-directed mutagenesis suffer, if anything, from the ability to produce more mutations than can be easily analyzed by electrophysiological techniques. The microbial aspects of S. cerevisiae offer the opportunity to greatly increase the efficiency of screening for functionally altered K+ channels. S. cerevisiae cells deleted for both TRK1 and TRK2 provide a desirable genetic background for investigating the effects of mutations in K+ channels since they can be assessed over a very broad functional range. For example, since the wild-type KAT1 K+ channel reduces the potassium requirement of trk1 delta trk2 delta cells from approximately 50 mM to less than 50 microM, the function of mutant channels can be assessed over a 1,000-fold range in concentration of the permeant ion. We have developed this system using a mutagenesis scheme that alters the amino acid sequence of the presumed pore region of KAT1. Regions of three amino acids in length can be saturated with substitutions and efficiently screened for function using this system. In addition, by testing the mutants for growth on media containing the appropriate competing ions, an in vivo indication of ion selectivity can be obtained. Saturation mutagenesis of the highly conserved GYG sequence in the channel pore reveals that few structural changes are tolerated if K+ selectivity is to be maintained. On the other hand, many of the mutants allow K+ permeation through the channel.
|Original language||English (US)|
|Number of pages||13|
|Journal||Symposia of the Society for Experimental Biology|
|State||Published - 1994|
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