Heterologous expression of K+ channels in XXXX : strategies for molecular analysis of structure and function.

@entity1522 @entity73206 @entity535 @entity73204 @entity972 @entity375 @entity73205
The ability to express heterologous proteins in K+ uptake-defective strains of @entity375 can be exploited to identify cDNAs encoding heterologous K+ channels. Moreover, the ability of heterologous @entity972 channels like @entity73204 and AKT1 to suppress completely the conditional negative growth phenotype of @entity375 cells containing mutations in @entity73205 or @entity73205 and @entity73206 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 @entity375 offer the opportunity to greatly increase the efficiency of screening for functionally altered K+ channels. @entity375 cells deleted for both @entity73205 and @entity73206 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 @entity73204 K+ channel reduces the @entity972 requirement of @entity73205 delta @entity73206 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 @entity535 sequence of the presumed pore region of @entity73204 . Regions of three @entity1522 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.
