Analysis of the molecular basis of calmodulin defects that affect ion channel-mediated cellular responses: site-specific mutagenesis and microinjection.
Analysis of the molecular basis of calmodulin defects that affect ion channel-mediated cellular responses: site-specific mutagenesis and microinjection.
复制标题
DOI：
10.1083/jcb.111.6.2537
复制
发表时间：
1990-12
期刊：
影响因子：
--
通讯作者：
Watterson DM
中科院分区：
文献类型：
--
作者：
Hinrichsen R;Wilson E;Lukas T;Craig T;Schultz J;Watterson DM
微注射钙调蛋白暂时恢复四片草履虫（cam1）钙调蛋白突变体离子通道介导的行为表型的能力依赖于残基101上存在的氨基酸侧链，即使在氨基酸序列的其余部分存在广泛的变化。对钙调蛋白中丝氨酸-101的保守分析表明，钙调蛋白调节这种离子通道相关细胞功能的能力可能是钙调蛋白在系统发育上广泛分布的生物学作用。通过体外位点特异性诱变和在大肠杆菌中表达产生了一系列仅在残基-101上不同的突变钙调素，纯化到化学均匀性，并测试了它们暂时恢复cam1 （pantophobiacA1）草履虫野生型行为表型的能力。含有甘氨酸-101或酪氨酸-101的钙调素活性最低；含有苯丙氨酸-101或丙氨酸-101的钙调素没有检测到活性。然而，作为比较的标准，所有的钙调素都能够激活一种钙调素调节的酶，肌球蛋白轻链激酶，这种酶对钙调素分子中其他地方的点突变很敏感。总的来说，这些结果支持了钙信号转导所需的钙调蛋白结构特征随被调节的特定途径而变化的假设，并提供了为什么钙调蛋白残基101的遗传突变在其表型效应中是非致命性和选择性的见解。
The ability of microinjected calmodulin to temporarily restore an ion channel-mediated behavioral phenotype of a calmodulin mutant in Paramecium tetraurelia (cam1) is dependent on the amino acid side chain that is present at residue 101, even when there is extensive variation in the rest of the amino acid sequence. Analysis of conservation of serine-101 in calmodulin suggests that the ability of calmodulin to regulate this ion channel-associated cell function may be a biological role of calmodulin that is widely distributed phylogenetically. A series of mutant calmodulins that differ only at residue-101 were produced by in vitro site-specific mutagenesis and expression in Escherichia coli, purified to chemical homogeneity, and tested for their ability to temporarily restore a wild-type behavioral phenotype to cam1 (pantophobiacA1) Paramecium. Calmodulins with glycine-101 or tyrosine-101 had minimal activity; calmodulins with phenylalanine-101 or alanine-101 had no detectable activity. However, as a standard of comparison, all of the calmodulins were able to activate a calmodulin- regulated enzyme, myosin light chain kinase, that is sensitive to point mutations elsewhere in the calmodulin molecule. Overall, these results support the hypothesis that the structural features of calmodulin required for the transduction of calcium signals varies with the particular pathway that is being regulated and provide insight into why inherited mutations of calmodulin at residue 101 are nonlethal and selective in their phenotypic effects.