Cornea Razvan L, Nitu Florentin, Gruber Simon, Kohler Katherine, Satzer Michael, Thomas David D, Fruen Bradley R
Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, 321 Church Street Southeast, Minneapolis, MN 55455, USA.
Proc Natl Acad Sci U S A. 2009 Apr 14;106(15):6128-33. doi: 10.1073/pnas.0813010106. Epub 2009 Mar 30.
Calmodulin (CaM) functions as a regulatory subunit of ryanodine receptor (RyR) channels, modulating channel activity in response to changing Ca(2+). To investigate the structural basis of CaM regulation of the RyR1 isoform, we used site-directed labeling of channel regulatory subunits and fluorescence resonance energy transfer (FRET). Donor fluorophore was targeted to the RyR1 cytoplasmic assembly by preincubating sarcoplasmic reticulum membranes with a fluorescent FK506-binding protein (FKBP), and FRET was determined following incubations in the presence of fluorescent CaMs in which acceptor fluorophore was attached within the N lobe, central linker, or C lobe. Results demonstrated strong FRET to acceptors attached within CaM's N lobe, whereas substantially weaker FRET was observed when acceptor was attached within CaM's central linker or C lobe. Surprisingly, Ca(2+) evoked little change in FRET to any of the 3 CaM domains. Donor-acceptor distances derived from our FRET measurements provide insights into CaM's location and orientation within the RyR1 3D architecture and the conformational switching that underlies CaM regulation of the channel. These results establish a powerful new approach to resolving the structure and function of RyR channels.
钙调蛋白（CaM）作为兰尼碱受体（RyR）通道的调节亚基，响应细胞内钙离子浓度（Ca(2+)）的变化来调节通道活性。为了研究CaM对RyR1亚型调节的结构基础，我们使用通道调节亚基的定点标记和荧光共振能量转移（FRET）技术。通过将肌浆网膜与荧光FK506结合蛋白（FKBP）预孵育，将供体荧光团靶向RyR1胞质组装体，然后在存在荧光CaM的情况下孵育后测定FRET，其中受体荧光团连接在N叶、中央连接区或C叶内。结果表明，与连接在CaM的N叶内的受体有很强的FRET，而当受体连接在CaM的中央连接区或C叶内时，观察到的FRET则明显较弱。令人惊讶的是，Ca(2+)对这3个CaM结构域的FRET几乎没有影响。从我们的FRET测量得出的供体-受体距离为了解CaM在RyR1三维结构中的位置和方向以及CaM对通道调节基础的构象转换提供了线索。这些结果建立了一种强有力的新方法来解析RyR通道的结构和功能。
