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

Plant Physiology and Biochemistry : PPB
Richard W MercierGerald A Berkowitz

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) en...Continue Reading

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Citations

Nov 23, 2010·Proceedings of the National Academy of Sciences of the United States of America·Zhi QiGerald A Berkowitz
Feb 27, 2007·FEBS Letters·Boaz KaplanHillel Fromm
Feb 15, 2005·Plant Physiology and Biochemistry : PPB·Geneviève EphritikhineNorbert Rolland
Dec 1, 2005·Journal of Experimental Botany·Rashid AliGerald A Berkowitz
Jan 21, 2021·International Journal of Molecular Sciences·Edwin Jarratt-BarnhamJulia M Davies

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