2B.E.E., Department of Pharmacology, Yale University School of Medicine, 333 Cedar St, New Haven, CT 06520-8066, USA.
FASEB J. 2014 May;28(5):2332-46. doi: 10.1096/fj.13-247106. Epub 2014 Feb 20.
Polycystin 2 (PC2) is a calcium-dependent calcium channel, and mutations to human PC2 (hPC2) are associated with polycystic kidney disease. The C-terminal tail of hPC2 contains 2 EF hand motifs, but only the second binds calcium. Here, we investigate whether these EF hand motifs serve as a calcium sensor responsible for the calcium dependence of PC2 function. Using NMR and bioinformatics, we show that the overall fold is highly conserved, but in evolutionarily earlier species, both EF hands bind calcium. To test whether the EF hand motif is truly a calcium sensor controlling PC2 channel function, we altered the number of calcium binding sites in hPC2. NMR studies confirmed that modified hPC2 binds an additional calcium ion. Single-channel recordings demonstrated a leftward shift in the calcium dependence, and imaging studies in cells showed that calcium transients were enhanced compared with wild-type hPC2. However, biophysics and functional studies showed that the first EF hand can only bind calcium and be functionally active if the second (native) calcium-binding EF hand is intact. These results suggest that the number and location of calcium-binding sites in the EF hand senses the concentration of calcium required for PC2 channel activity and cellular function.
多囊蛋白 2（PC2）是一种钙离子依赖的钙离子通道，人类 PC2（hPC2）的突变与多囊肾病有关。hPC2 的 C 端尾部包含 2 个 EF 手模体，但只有第二个结合钙离子。在这里，我们研究这些 EF 手模体是否作为一个钙离子感受器，负责 PC2 功能的钙离子依赖性。通过 NMR 和生物信息学，我们表明整体折叠高度保守，但在进化早期的物种中，两个 EF 手都结合钙离子。为了测试 EF 手模体是否真的是一个控制 PC2 通道功能的钙离子感受器，我们改变了 hPC2 中钙结合位点的数量。NMR 研究证实，修饰后的 hPC2 结合了额外的钙离子。单通道记录显示钙离子依赖性向左移动，细胞内成像研究表明与野生型 hPC2 相比，钙瞬变增强。然而，生物物理学和功能研究表明，如果第二个（天然）钙结合 EF 手保持完整，第一个 EF 手只能结合钙离子并具有功能活性。这些结果表明，EF 手的钙结合位点的数量和位置可以感知 PC2 通道活性和细胞功能所需的钙离子浓度。
