Direct visualization of interaction between calmodulin and connexin45.
Direct visualization of interaction between calmodulin and connexin45.
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DOI：
10.1042/bcj20170426
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发表时间：
2017-11-27
期刊：
影响因子：
--
通讯作者：
Yang JJ
中科院分区：
文献类型：
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作者：
Zou J;Salarian M;Chen Y;Zhuo Y;Brown NE;Hepler JR;Yang JJ
钙调素（CaM）是一种细胞内Ca ~（2+）转导蛋白，在广泛的Ca ~（2+）信号网络中参与多种活动。以前的研究表明，Ca 2 +/CaM复合物可能参与间隙连接调节通过相互作用与假定的钙调素结合基序的连接蛋白，然而，CaM和连接蛋白之间的直接相互作用的证据仍然难以捉摸的日期，由于与膜蛋白的研究的挑战。在此，我们首次报道了在生理条件下，在活细胞中通过生物发光共振能量转移（bioluminescence resonance energy transfer，BLS）检测钙调素（CaM）与γ-连接蛋白45（connexin 45）的直接相互作用。细胞内CaM和Cx45之间的相互作用强烈依赖于细胞内Ca 2+浓度，并且可以被CaM抑制剂N-（6-氨基己基）-5-氯-1-萘磺酰胺盐酸盐（W7）阻断。我们进一步揭示了钙调素结合位点的胞质环（残基164-186）的Cx45使用肽模型。通过荧光标记的CaM监测，发现CaM和Cx45肽之间观察到的强结合（Kd ~ 5 nM）是Ca 2+依赖性的。此外，高分辨率核磁共振光谱表明，钙调素和Cx45肽结合导致全球化学位移的变化15 N-标记的钙调素，但不改变结构的大小。涉及CaM的N-和C-结构域与Cx45肽相互作用的观察结果不同于与来自另一连接蛋白家族的Cx 50的相互作用。这种相互作用进一步增加了CaM的Ca 2+敏感性，特别是在N-末端结构域。本研究的结果表明，螺旋度和相互作用模式的胞质环可能有助于钙调素的连接蛋白的调制。
Calmodulin (CaM) is an intracellular Ca2+ transducer involved in numerous activities in a broad Ca2+ signaling network. Previous studies have suggested that the Ca2+/CaM complex may participate in gap junction regulation via interaction with putative CaM-binding motifs in connexins; however, evidence of direct interactions between CaM and connexins has remained elusive to date due to challenges related to the study of membrane proteins. Here, we report the first direct interaction of CaM with Cx45 (connexin45) of γ-family in living cells under physiological conditions by monitoring bioluminescence resonance energy transfer. The interaction between CaM and Cx45 in cells is strongly dependent on intracellular Ca2+ concentration and can be blocked by the CaM inhibitor, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W7). We further reveal a CaM-binding site at the cytosolic loop (residues 164–186) of Cx45 using a peptide model. The strong binding (Kd ~ 5 nM) observed between CaM and Cx45 peptide, monitored by fluorescence-labeled CaM, is found to be Ca2+-dependent. Furthermore, high-resolution nuclear magnetic resonance spectroscopy reveals that CaM and Cx45 peptide binding leads to global chemical shift changes of 15N-labeled CaM, but does not alter the size of the structure. Observations involving both N- and C-domains of CaM to interact with the Cx45 peptide differ from the embraced interaction with Cx50 from another connexin family. Such interaction further increases Ca2+ sensitivity of CaM, especially at the N-terminal domain. Results of the present study suggest that both helicity and the interaction mode of the cytosolic loop are likely to contribute to CaM’s modulation of connexins.