Yeast hygromycin sensitivity as a functional assay of cyclic nucleotide gated cation channels.
Cyclic nucleotide gated cation channels (CNGCs) are a large (20 genes in Arabidopsis thaliana) family of plant ligand gated (i.e. cyclic nucleotides activate currents) ion channels, however, little is known about their functional properties. One reason for this is the recalcitrance of plant CNGC expression in heterologous systems amenable to patch clamp studies. Here, we show results demonstrating the efficacy of using growth of a K+ uptake-deficient yeast (trk1,2) as a functional assay of CNGCs as inwardly-conducting cell membrane cation (K+) transporters. Prior work demonstrated that trk1,2 is hypersensitive to the antibiotic hygromycin (hyg) and that expression of an inwardly conducting K+ transporter suppresses hyg hypersensitivity. We find that increasing [hyg] in solid YPD medium inhibits trk1,2 growth around a filter disk saturated with 3 M K+. Northern analysis indicated that message is transcribed in trk1,2 transformed with the CNGC coding sequences. Confocal imaging of yeast expressing CNGC-fluorescent fusion proteins indicated channel targeting to the cell membrane. Trk1,2 expressing several plant CNGCs grown in the presence of hyg demonstrated (a) greater growth than trk1,2 transformed with empty plasmid, and (b) enhanced growth when cAMP was added to the medium. Alternatively, cAMP inhibited growth of yeast transformed with either the empty plasmid, or the plant K+ channel KAT1; this channel is not a CNGC. Growth of trk1,2 was dependent on filter disk [K+]; suggesting that complementation of hyg hypersensitivity due to presence of a functional plant CNGC was dependent on K+ movement into the cytosol. We conclude that plant CNGC functional characterization can be facilitated by this assay system.
Mercier
Richard W
RW
Agricultural Biotechnology Laboratory, Department of Plant Science, University of Connecticut, 1390 Storrs Road, U-4163, Storrs, CT 06269-4163, USA.
Rabinowitz
Natasha M
NM
Ali
Rashid
R
Gaxiola
Roberto A
RA
Berkowitz
Gerald A
GA
D017360:Arabidopsis / Q000235:genetics / Q000502:physiology; D029681:Arabidopsis Proteins / Q000235:genetics / Q000502:physiology; D003994:Bucladesine / Q000494:pharmacology; D054815:Cyclic Nucleotide-Gated Cation Channels; D007473:Ion Channels / Q000235:genetics / Q000502:physiology*; D010957:Plasmids / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000502:physiology*; D014158:Transcription, Genetic
D029681:Arabidopsis Proteins; D054815:Cyclic Nucleotide-Gated Cation Channels; D007473:Ion Channels; D003994:Bucladesine