TY - JOUR
T1 - Yeast hygromycin sensitivity as a functional assay of cyclic nucleotide gated cation channels
AU - Mercier, Richard W.
AU - Rabinowitz, Natasha M.
AU - Ali, Rashid
AU - Gaxiola, Roberto A.
AU - Berkowitz, Gerald A.
N1 - Funding Information:
This work was supported by the National Science Foundation (award no. MCB-0344141) and the USDA National Research Initiative (award no. 2001-35304-10927). We thank Dr. Gerald Fink (Massachusetts Institute of Technology, Cambridge, MA) for the gift of the ACT1 probe, and Dr. Susan Michaelis (Johns Hopkins University, Baltimore, MD) for the gift of the pSM1052 plasmid.
Copyright:
Copyright 2008 Elsevier B.V., All rights reserved.
PY - 2004/6
Y1 - 2004/6
N2 - 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.
AB - 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.
KW - CNGC
KW - E
KW - GFP
KW - HEK
KW - Ion channel
KW - Plant cyclic nucleotide gated cation channels
KW - YNB
KW - YPD
KW - cAMP
KW - cyclic nucleotide gated cation channel
KW - green fluorescent protein
KW - human embryonic kidney
KW - hyg
KW - hygromycin B
KW - membrane potential
KW - yeast nitrogen base
UR - http://www.scopus.com/inward/record.url?scp=3142570823&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=3142570823&partnerID=8YFLogxK
U2 - 10.1016/j.plaphy.2004.04.007
DO - 10.1016/j.plaphy.2004.04.007
M3 - Article
C2 - 15246066
AN - SCOPUS:3142570823
SN - 0981-9428
VL - 42
SP - 529
EP - 536
JO - Plant Physiology and Biochemistry
JF - Plant Physiology and Biochemistry
IS - 6
ER -