Analysis of the mKir2.1 channel activity in potassium influx defective Saccharomyces cerevisiae strains determined as changes in growth characteristics.
Potassium uptake defective Saccharomyces cerevisiae strains (Deltatrk1,2 and Deltatrk1,2 Deltatok1) were used for the phenotypic analysis of the mouse inward rectifying Kir2.1 channel by growth analysis. Functional expression of both, multi-copy plasmid and chromosomally expressed GFP-mKir2.1 fusion constructs complemented the potassium uptake deficient phenotype in a pHout dependent manner. Upon application of Hygromycin B to chromosomally mKir2.1 expressing cells, significantly lower toxin sensitivity (EC50 15.4 microM) compared to Deltatrk1,2 Deltatok1 cells (EC50 2.6 microM) was observed. Growth determination of mKir2.1 expressing strains upon application of Ag+, Cs+ and Ba2+ as known blockers of mKir2.1 channels revealed significantly decreased channel function. Cells with mKir2.1 were about double sensitive to AgNO3, 350-fold more sensitive to CsCl and 1500-fold more sensitive to BaCl2 in comparison to the respective controls indicating functional expression and correct pharmacology.
Hasenbrink
Guido
G
Molecular Bioenergetics, IZMB, Universität Bonn, Kirschallee 1, 53115 Bonn, Germany.
Schwarzer
Sarah
S
Kolacna
Lucie
L
Ludwig
Jost
J
Sychrova
Hana
H
Lichtenberg-Fraté
Hella
H
D000818:Animals; D027682:Cation Transport Proteins / Q000235:genetics; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005816:Genetic Complementation Test; D006921:Hygromycin B / Q000494:pharmacology; D017136:Ion Transport / Q000235:genetics / Q000502:physiology; D051379:Mice; D009154:Mutation / Q000235:genetics; D011188:Potassium / Q000032:analysis / Q000378:metabolism*; D026902:Potassium Channel Blockers / Q000494:pharmacology*; D024661:Potassium Channels, Inwardly Rectifying / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics* / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics
D027682:Cation Transport Proteins; C489461:Kir2.1 channel; D026902:Potassium Channel Blockers; D024661:Potassium Channels, Inwardly Rectifying; D029701:Saccharomyces cerevisiae Proteins; C085258:TRK2 protein, S cerevisiae; C068664:TRK1 protein, S cerevisiae; D006921:Hygromycin B; D011188:Potassium