Uozumi N, Gassmann W, Cao Y, Schroeder J I
Department of Biology, University of California, San Diego, La Jolla 92093-0116, USA.
J Biol Chem. 1995 Oct 13;270(41):24276-81. doi: 10.1074/jbc.270.41.24276.
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 (K1/2 = 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.
拟南芥cDNA，即KAT1，编码一种超极化激活的钾离子通道。在本研究中，我们结合随机定点诱变、在钾吸收缺陷型酵母菌株中的遗传筛选以及非洲爪蟾卵母细胞中的电生理分析，来鉴定内向整流钾离子通道KAT1在阳离子选择性方面的显著改变。第256位的苏氨酸被其他11种氨基酸残基取代。其中6个突变的KAT1 cDNA在低钾浓度下可互补钾吸收缺陷型酵母菌株。在这些突变体中，两个突变体（T256D和T256G）显示出酵母生长对高铵浓度的敏感性，并且相对于钾离子，铷离子和铵离子的电流幅度显著增加了39 - 72倍。这些单点突变产生了对铷离子和铵离子有选择性的通道，其铷离子和铵离子电流的幅度比钾离子电流大约大10 - 13倍，而野生型KAT1的铵离子与钾离子电流幅度比为0.28。这种阳离子特异性的强烈转变且不丧失一般选择性，超过了报道的电压依赖性钾离子通道孔域中其他突变的情况。在此位点的另外两个突变体（T256E和T256Q）中，酵母生长受到钠的严重损害，它们被毫摩尔级的钠所阻断（T256E的K1/2 = 1.1 mM），尽管野生型通道不受110 mM钠的阻断。有趣的是，酵母在有毒阳离子存在下生长的能力与KAT1突变体的生物物理特性相关，这说明了在酵母中进行定性钾离子通道突变体筛选的潜力。这些数据表明，KAT1中第256位氨基酸侧链的大小对于阳离子通透至关重要，并且该位点在决定超极化激活钾离子通道的阳离子选择性方面起着关键作用。
