Departments of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520; Departments of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520.
Departments of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520.
J Biol Chem. 2012 May 18;287(21):17232-17240. doi: 10.1074/jbc.M112.354613. Epub 2012 Apr 3.
Polycystin-2 (PC2) is a Ca(2+)-permeable transient receptor potential channel activated and regulated by changes in cytoplasmic Ca(2+). PC2 mutations are responsible for ∼15% of autosomal dominant polycystic kidney disease. Although the C-terminal cytoplasmic tail of PC2 has been shown to contain a Ca(2+)-binding EF-hand domain, the molecular basis of PC2 channel gating by Ca(2+) remains unknown. We propose that the PC2 EF-hand is a Ca(2+) sensor required for channel gating. Consistent with this, Ca(2+) binding causes a dramatic decrease in the radius of gyration (R(g)) of the PC2 EF-hand by small angle x-ray scattering and significant conformational changes by NMR. Furthermore, increasing Ca(2+) concentrations cause the C-terminal cytoplasmic tail to transition from a mixture of extended oligomers to a single compact dimer by analytical ultracentrifugation, coupled with a >30 Å decrease in maximum interatomic distance (D(max)) by small angle x-ray scattering. Mutant PC2 channels unable to bind Ca(2+) via the EF-hand are inactive in single-channel planar lipid bilayers and inhibit Ca(2+) release from ER stores upon overexpression in cells, suggesting dominant negative properties. Our results support a model where PC2 channels are gated by discrete conformational changes in the C-terminal cytoplasmic tail in response to changes in cytoplasmic Ca(2+) levels. These properties of PC2 are lost in autosomal dominant polycystic kidney disease, emphasizing the importance of PC2 to kidney cell function. We speculate that PC2 and the Ca(2+)-dependent transient receptor potential channels in general are regulated by similar conformational changes in their cytoplasmic domains that are propagated to the channel pore.
多囊蛋白-2（PC2）是一种钙通透型瞬时受体电位通道，其活性受细胞质钙离子浓度变化的调节。PC2 突变导致约 15%的常染色体显性多囊肾病。虽然已经证明 PC2 的胞质尾部含有一个钙结合 EF 手结构域，但 PC2 通道对钙离子的门控的分子基础仍不清楚。我们提出 PC2 的 EF 手是钙传感器，是通道门控所必需的。与这一观点一致的是，钙结合导致 PC2 EF 手的回旋半径（R(g)）通过小角 X 射线散射显著减小，并通过 NMR 产生显著的构象变化。此外，增加钙离子浓度导致分析超速离心时，胞质尾部的 C 端从延伸的寡聚物混合物转变为单一的紧凑二聚体，同时通过小角 X 射线散射，最大原子间距离（D(max)）减小了>30 Å。不能通过 EF 手结合钙离子的突变 PC2 通道在单通道平面脂质双层中是无活性的，并且在细胞中过表达时抑制内质网库中的钙释放，表明具有显性负性特性。我们的结果支持这样一种模型，即 PC2 通道通过 C 端胞质尾部的离散构象变化来响应细胞质钙离子浓度的变化来进行门控。这些特性在常染色体显性多囊肾病中丢失，强调了 PC2 对肾脏细胞功能的重要性。我们推测 PC2 和一般的钙离子依赖性瞬时受体电位通道通过其胞质域中的类似构象变化来调节，这些构象变化被传递到通道孔。
