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首页> 外文期刊>European Journal of Medicinal Chemistry: Chimie Therapeutique >Yeast hygromycin sensitivity as a functional assay of cyclic nucleotide gated cation channels
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Yeast hygromycin sensitivity as a functional assay of cyclic nucleotide gated cation channels

机译：酵母潮霉素敏感性作为功能性测定环核苷酸门控阳离子通道

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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.
机译：环状核苷酸门控阳离子通道（CNGC）是植物配体门控（即环状核苷酸激活电流）离子通道的大家族（拟南芥中有20个基因），然而，对其功能特性知之甚少。其原因之一是在适于膜片钳研究的异源系统中植物CNGC表达的顽固性。在这里，我们显示的结果证明了使用K +摄取缺陷型酵母（trk1,2）的生长作为CNGC作为内向传导性细胞膜阳离子（K +）转运蛋白的功能测定的功效。先前的研究表明，trk1,2对抗生素潮霉素（hyg）过敏，向内传导的K +转运蛋白的表达抑制了hyg过敏。我们发现，在固体YPD培养基中增加[hyg]会抑制trk1,2在3 M K〜+饱和的滤盘周围的生长。 Northern分析表明消息是在用CNGC编码序列转化的trk1,2中转录的。表达CNGC荧光融合蛋白的酵母的共聚焦成像表明通道靶向细胞膜。表达在hyg存在下生长的几种植物CNGC的Trk1,2表现出（a）比用空质粒转化的trk1,2更大的生长，并且（b）当将cAMP添加到培养基中时，生长增强。另外，cAMP抑制了用空质粒或植物K +通道KAT1转化的酵母的生长。此频道不是CNGC。 trk1,2的生长取决于滤盘[K +];提示由于功能性植物CNGC的存在，hyg过敏反应的补充取决于K +向细胞质中的移动。我们得出结论，该测定系统可促进植物CNGC功能表征。

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