title: Expression of plant cyclic nucleotide-gated cation channels in yeast.
abstract: The functional properties of inwardly conducting plant cyclic nucleotide-gated cation channels (CNGCs) have not been thoroughly characterized due in part to the recalcitrance of their functional expression in heterologous systems. Here, K+ uptake-deficient mutants of yeast (trk1,2) and Escherichia coli (LB650), as well as the Ca2+-uptake yeast mutant mid1,cch1, were used for functional characterization of Arabidopsis thaliana CNGCs, with the aim of identifying some of the cultural and physiological conditions that impact on plant CNGC function in heterologous systems. Use of the Ca2+-uptake yeast mutant provided the first evidence consistent with Ca2+ conduction by the A. thaliana CNGC AtCNGC1. Expression of AtCNGC1 in LB650 demonstrated that mutants of Escherichia coli (which has no endogenous calmodulin) can also be used to study functional properties of CNGCs. Expression of AtCNGC2 and AtCNGC4 enhanced growth of trk1,2 in the presence of hygromycin; AtCNGC1 has less of an effect. Deletion of the AtCNGC1 calmodulin-binding domain enhanced growth of trk1,2 at low external K+ but not of LB650, suggesting that yeast calmodulin may bind to, and down-regulate this plant channel. In vitro binding studies confirmed this physical interaction. Northern analysis, green fluorescent protein:AtCNGC1 fusion protein expression, as well as an antibody raised against a portion of AtCNGC1, were used to monitor expression of AtCNGC1 and deletion constructs of the channel in the heterologous systems. In the presence of the activating ligand cAMP, expression of the AtCNGC1 channel with the calmodulin-binding domain deleted increased intracellular [K+] of trk1,2. Trk1,2 is hypersensitive to the toxic cations spermine, tetramethylamine, and NH4+. These compounds, as well as amiloride, inhibited trk1,2 growth and thereby improved the efficacy of this yeast mutant as a heterologous expression system for CNGCs. In addition to characterizing mutants of yeast and E. coli as assay systems for plant CNGCs, work presented in this report demonstrates, for the first time, that a plant CNGC can retain ion channel function despite (partial) deletion of its calmodulin-binding domain and that yeast calmodulin can bind to and possibly down-regulate a plant CNGC.
authors.affiliation: Agricultural Biotechnology Laboratory, Department of Plant Science, University of Connecticut, U-4067 Storrs Road, Storrs, CT 06269-4067, USA.
authors.forename: Rashid
authors.initials: R
authors.lastname: Ali
authors.forename: Raymond E
authors.initials: RE
authors.lastname: Zielinski
authors.forename: Gerald A
authors.initials: GA
authors.lastname: Berkowitz
mesh_terms: D017360:Arabidopsis / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000096:biosynthesis / Q000235:genetics*; D003001:Cloning, Molecular; D003470:Culture Media / Q000737:chemistry; D054815:Cyclic Nucleotide-Gated Cation Channels; D004926:Escherichia coli / Q000235:genetics; D005816:Genetic Complementation Test; D007473:Ion Channels / Q000096:biosynthesis / Q000235:genetics*; D009154:Mutation; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014170:Transformation, Genetic
chemical_list: D029681:Arabidopsis Proteins; D003470:Culture Media; D054815:Cyclic Nucleotide-Gated Cation Channels; D007473:Ion Channels