Identification of Strong Modifications in Cation Selectivity in an Arabidopsis Inward Rectifying Potassium Channel by Mutant Selection in Yeast (*)
Identification of Strong Modifications in Cation Selectivity in an Arabidopsis Inward Rectifying Potassium Channel by Mutant Selection in Yeast (*)
复制标题
通过酵母突变体选择鉴定拟南芥内向整流钾通道中阳离子选择性的强修饰 (*)
DOI：
10.1074/jbc.270.41.24276
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
J. Schroeder
中科院分区：
文献类型：
--
作者：
N. Uozumi;W. Gassmann;Yongwei Cao;J. Schroeder
拟南芥（Arabidopsis thaliana） cDNA KAT1编码一个超极化激活的K+通道。在本研究中，我们利用随机位点定向诱变、钾摄取缺陷酵母菌株的遗传筛选和非洲爪蟾卵母细胞的电生理分析相结合，确定了向内纠偏K+通道KAT1的阳离子选择性的强修饰。第256位的苏氨酸被另外11个氨基酸残基取代。这些突变的KAT1 cdna中的6个在低钾浓度下补充了K+摄取不足的酵母菌株。其中，两个突变体（T256D和T256G）表现出对高铵浓度的敏感，铷和铵离子的电流幅度相对于K+急剧增加了39-72倍。这些单位点突变产生Rb+和NH4+选择性通道，其Rb+和NH4+电流的振幅约为K+电流的10-13倍，而野生型KAT1的NH4+与K+电流的振幅比为0.28。这种阳离子特异性的强转换而不丧失一般选择性，超过了在电压依赖性K+通道的孔域中其他突变的报道。在这一位点的另外两个突变体（T256E和T256Q）中，钠极大地损害了酵母的生长，它们被毫摩尔钠（T256E的K = 1.1 mM）阻断，尽管野生型通道没有被110 mM钠阻断。有趣的是，酵母在有毒阳离子存在下的生长能力与KAT1突变体的生物物理特性相关，这说明了酵母中K+通道突变体的定性选择潜力。这些数据表明，KAT1中256位氨基酸侧链的大小对于阳离子渗透很重要，并且该位点在决定超极化活化钾通道的阳离子选择性方面起着至关重要的作用。
The Arabidopsis thaliana cDNA, KAT1, encodes a hyperpolarization-activated K+ channel. In the present study, we utilized a combination of random site-directed mutagenesis, genetic screening in a potassium uptake-deficient yeast strain, and electrophysiological analysis in Xenopus oocytes to identify strong modifications in cation selectivity of the inward rectifying K+ channel KAT1. Threonine at position 256 was replaced by 11 other amino acid residues. Six of these mutated KAT1 cDNAs complemented a K+ uptake-deficient yeast strain at low concentrations of potassium. Among these, two mutants (T256D and T256G) showed a sensitivity of yeast growth toward high ammonium concentrations and a dramatic increase in current amplitudes of rubidium and ammonium ions relative to K+ by 39-72-fold. These single site mutations gave rise to Rb+- and NH4+-selective channels with Rb+ and NH4+ currents that were approximately 10-13-fold greater in amplitude than K+ currents, whereas the NH4+ to K+ current amplitude ratio of wild type KAT1 was 0.28. This strong conversion in cation specificity without loss of general selectivity exceeds those reported for other mutations in the pore domain of voltage-dependent K+ channels. Yeast growth was greatly impaired by sodium in two other mutants at this site (T256E and T256Q), which were blocked by millimolar sodium (K = 1.1 mM for T256E), although the wild type channel was not blocked by 110 mM sodium. Interestingly, the ability of yeast to grow in the presence of toxic cations correlated to biophysical properties of KAT1 mutants, illustrating the potential for qualitative K+ channel mutant selection in yeast. These data suggest that the size of the side chain of the amino acid at position 256 in KAT1 is important for enabling cation permeation and that this site plays a crucial role in determining the cation selectivity of hyperpolarization-activated potassium channels.
影响因子：
56.9
作者：
HEGINBOTHAM, L;ABRAMSON, T;MACKINNON, R
通讯作者：
MACKINNON, R