Bian S, Favre I, Moczydlowski E
Departments of Pharmacology and Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06520-8066, USA.
Proc Natl Acad Sci U S A. 2001 Apr 10;98(8):4776-81. doi: 10.1073/pnas.081072398. Epub 2001 Mar 27.
Mutational and biophysical analysis suggests that an intracellular COOH-terminal domain of the large conductance Ca(2+)-activated K(+) channel (BK channel) contains Ca(2+)-binding site(s) that are allosterically coupled to channel opening. However the structural basis of Ca(2+) binding to BK channels is unknown. To pursue this question, we overexpressed the COOH-terminal 280 residues of the Drosophila slowpoke BK channel (Dslo-C280) as a FLAG- and His(6)-tagged protein in Escherichia coli. We purified Dslo-C280 in soluble form and used a (45)Ca(2+)-overlay protein blot assay to detect Ca(2+) binding. Dslo-C280 exhibits specific binding of (45)Ca(2+) in comparison with various control proteins and known EF-hand Ca(2+)-binding proteins. A mutation (D5N5) of Dslo-C280, in which five consecutive Asp residues of the "Ca-bowl" motif are changed to Asn, reduces (45)Ca(2+)-binding activity by 56%. By electrophysiological assay, the corresponding D5N5 mutant of the Drosophila BK channel expressed in HEK293 cells exhibits lower Ca(2+) sensitivity for activation and a shift of approximately +80 mV in the midpoint voltage for activation. This effect is associated with a decrease in the Hill coefficient (N) for activation by Ca(2+) and a reduction in apparent Ca(2+) affinity, suggesting the loss of one Ca(2+)-binding site per monomer. These results demonstrate a functional correlation between Ca(2+) binding to a specific region of the BK protein and Ca(2+)-dependent activation, thus providing a biochemical approach to study this process.
突变和生物物理分析表明，大电导钙激活钾通道（BK通道）的细胞内羧基末端结构域含有与通道开放变构偶联的钙结合位点。然而，钙与BK通道结合的结构基础尚不清楚。为了探究这个问题，我们在大肠杆菌中过表达了果蝇慢poke BK通道（Dslo-C280）的羧基末端280个残基，作为一种带有FLAG和His(6)标签的蛋白质。我们以可溶形式纯化了Dslo-C280，并使用45Ca(2+)覆盖蛋白印迹分析来检测钙结合。与各种对照蛋白和已知的EF手型钙结合蛋白相比，Dslo-C280表现出对45Ca(2+)的特异性结合。Dslo-C280的一个突变（D5N5），即“钙碗”基序中的五个连续天冬氨酸残基被突变为天冬酰胺，使45Ca(2+)结合活性降低了56%。通过电生理分析，在HEK293细胞中表达的果蝇BK通道的相应D5N5突变体对钙激活的敏感性较低，激活中点电压偏移约+80 mV。这种效应与钙激活的希尔系数（N）降低以及表观钙亲和力降低有关，表明每个单体失去了一个钙结合位点。这些结果证明了钙与BK蛋白特定区域的结合与钙依赖性激活之间的功能相关性，从而为研究这一过程提供了一种生化方法。
