Studying ion channels using yeast genetics.
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Animals↗
Biomedical subjects
R L Nakamura
Publications and source records attributed to R L Nakamura.
Determination of key structural requirements of a K+ channel pore.
Among the highly conserved sites in K+ channel pores, the tyrosine-glycine sequence is believed to play an important role in selectivity. Here we describe a novel approach in which comprehensive mutagenesis of the YG sites of the voltage-gated K+ channel, Kat1, is combined with phenotypic screening in Saccharomyces cerevisiae and electrophysiological analysis in Xenopus oocytes to determine the roles of these sites in K+ selectivity. We show that structural constraints necessitate a tyrosine or phenylalanine at the first position to confer full K+ selectivity. Substitution to arginine creates a channel titratable by external pH, suggesting that the side group at this position may line the channel pore. Permeation is abolished by any increase in bulk at the adjacent glycine position unless accompanied by a compensatory mutation at the tyrosine site. These results suggest a model in which the selectivity filter of the K+ channel requires an aromatic residue paired with glycine within the pore loop in order to maintain maximal K+ selectivity.
Amino Acid Sequence↗
Expression of an Arabidopsis potassium channel gene in guard cells.
The Arabidopsis thaliana KAT1 cDNA encodes a voltage-gated inward-rectifying K+ channel. A KAT1 genomic DNA clone was isolated and sequenced, and a 5' promoter and coding sequences containing eight introns were identified. Reporter gene analysis of transgenic plants containing the KAT1 promoter fused to bacterial beta-glucuronidase showed robust beta-glucuronidase activity primarily in guard cells.
Arabidopsis↗
Heterologous expression of K+ channels in Saccharomyces cerevisiae: strategies for molecular analysis of structure and function.
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.
Amino Acid Sequence↗