Gangopadhyay Jaya P, Ikemoto Noriaki
Boston Biomedical Research Institute, Watertown, MA 02472, USA.
Biochem J. 2008 Apr 15;411(2):415-23. doi: 10.1042/BJ20071375.
In the present study we show that the interaction of the CaM (calmodulin)-binding domain (Lys(3614)-Asn(3643)) with the Cys(4114)-Asn(4142) region (a region included in the CaM-like domain) serves as an intrinsic regulator of the RyR1 (type-1 ryanodine receptor). We tested the effects of antibodies raised against the two putative key regions of RyR1 [anti-(Lys(3614)-Asn(3643)) and anti-(Cys(4114)-Asn(4142)) antibodies]. Both antibodies produced significant inhibition of [3H]ryanodine-binding activity of RyR1. This suggests that the inter-domain interaction between the two domains, Lys(3614)-Asn(3643) and Cys(4114)-Asn(4142), activates the channel, and that the binding of antibody to either side of the interacting domain pair interfered with the formation of a 'channel-activation link' between the two regions. In order to spectroscopically monitor the mode of interaction of these domains, the site of inter-domain interaction was fluorescently labelled with MCA [(7-methoxycoumarin-4-yl)acetyl] in a site-directed manner. The accessibility of the bound MCA to a large molecular mass fluorescence quencher, BSA-QSY (namely, the size of a gap between the interacting domains) decreased with an increase of [Ca2+] in a range of 0.03-2.0 microM, as determined by Stern-Volmer fluorescence quenching analysis. The Ca2+-dependent decrease in the quencher accessibility was more pronounced in the presence of 150 microM 4-CmC (4-chlorometacresol), and was reversed by 1 mM Mg2+ (a well-known inhibitor of Ca2+/agonist-induced channel activation). These results suggest that the Lys(3614)-Asn(3643) and Cys(4114)-Asn(4142) regions of RyR1 interact with each other in a Ca2+- and agonist-dependent manner, and this serves as a mechanism of Ca2+- and agonist-dependent activation of the RyR1 Ca2+ channel.
在本研究中，我们表明钙调蛋白（CaM）结合结构域（赖氨酸（3614）-天冬酰胺（3643））与半胱氨酸（4114）-天冬酰胺（4142）区域（钙调蛋白样结构域中的一个区域）之间的相互作用充当1型兰尼碱受体（RyR1）的内在调节因子。我们测试了针对RyR1两个假定关键区域产生的抗体的作用[抗（赖氨酸（3614）-天冬酰胺（3643））抗体和抗（半胱氨酸（4114）-天冬酰胺（4142））抗体]。两种抗体均对RyR1的[3H]兰尼碱结合活性产生了显著抑制作用。这表明赖氨酸（3614）-天冬酰胺（3643）和半胱氨酸（4114）-天冬酰胺（4142）这两个结构域之间的结构域间相互作用激活了通道，并且抗体与相互作用结构域对的任一侧结合都会干扰两个区域之间“通道激活连接”的形成。为了通过光谱法监测这些结构域的相互作用模式，以定点方式用MCA [（7-甲氧基香豆素-4-基）乙酰基]对结构域间相互作用位点进行荧光标记。通过斯特恩-沃尔默荧光猝灭分析确定，在0.03-2.0微摩尔范围内，随着[Ca2+]的增加，结合的MCA对大分子质量荧光猝灭剂牛血清白蛋白-QSY的可及性（即相互作用结构域之间间隙的大小）降低。在存在150微摩尔4-氯间甲酚（4-CmC）的情况下，猝灭剂可及性的Ca2+依赖性降低更为明显，并且被1毫摩尔Mg2+（一种众所周知的Ca2+/激动剂诱导的通道激活抑制剂）逆转。这些结果表明，RyR1的赖氨酸（3614）-天冬酰胺（3643）和半胱氨酸（4114）-天冬酰胺（4142）区域以Ca2+和激动剂依赖性方式相互作用，这充当了Ca2+和激动剂依赖性激活RyR1 Ca2+通道的一种机制。
