Yeast hygromycin sensitivity as a functional assay of cyclic nucleotide gated cation channels

Richard W. Mercier, Natasha M. Rabinowitz, Rashid Ali, Roberto Gaxiola, Gerald A. Berkowitz

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)529-536
Number of pages8
JournalPlant Physiology and Biochemistry
Volume42
Issue number6
DOIs
StatePublished - Jun 2004
Externally publishedYes

Fingerprint

Cyclic Nucleotide-Gated Cation Channels
cyclic nucleotides
Yeast
Assays
cations
Yeasts
yeasts
assays
Growth
Cell membranes
hypersensitivity
Hypersensitivity
Plasmids
cell membranes
transporters
plasmids
Cell Membrane
hygromycin A
Channel coding
Cyclic Nucleotides

Keywords

  • cAMP
  • CNGC
  • cyclic nucleotide gated cation channel
  • E
  • GFP
  • green fluorescent protein
  • HEK
  • human embryonic kidney
  • hyg
  • hygromycin B
  • Ion channel
  • membrane potential
  • Plant cyclic nucleotide gated cation channels
  • yeast nitrogen base
  • YNB
  • YPD

ASJC Scopus subject areas

  • Plant Science
  • Biochemistry
  • Biotechnology

Cite this

Yeast hygromycin sensitivity as a functional assay of cyclic nucleotide gated cation channels. / Mercier, Richard W.; Rabinowitz, Natasha M.; Ali, Rashid; Gaxiola, Roberto; Berkowitz, Gerald A.

In: Plant Physiology and Biochemistry, Vol. 42, No. 6, 06.2004, p. 529-536.

Research output: Contribution to journalArticle

Mercier, Richard W. ; Rabinowitz, Natasha M. ; Ali, Rashid ; Gaxiola, Roberto ; Berkowitz, Gerald A. / Yeast hygromycin sensitivity as a functional assay of cyclic nucleotide gated cation channels. In: Plant Physiology and Biochemistry. 2004 ; Vol. 42, No. 6. pp. 529-536.
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KW - yeast nitrogen base

KW - YNB

KW - YPD

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