Chiang Wen, Byrem Todd, Zhang Haiyan, Strasburg Gale
Department of Food Science and Human Nutrition, Michigan State University, R3365 Anthony Hall, East Lansing, MI 48824, USA.
J Muscle Res Cell Motil. 2007;28(1):59-66. doi: 10.1007/s10974-007-9106-9. Epub 2007 May 16.
Ca(2+) release during excitation-contraction coupling in avian skeletal muscle is controlled by two ryanodine receptor isoforms, alphaRYR and betaRYR. Two other proteins, dihydropyridine receptor (DHPR) and calmodulin (CaM), have been shown to play important roles in regulating the RYR channel activity. In the current study, we measured the protein contents of DHPR and RYR in turkey skeletal muscle and obtained a ratio of 1:1 between DHPR and alphaRYR which suggests that only a subpopulation of alphaRYR is interacting with DHPR. Two CaM derivatives, the photoactivable crosslinking probe [(125)I]-Bz-CaM and metabolically labeled probe [(35)S]CaM, were used to study the interaction between CaM and RYR isoforms in turkey skeletal muscle. The alphaRYR and betaRYR displayed a marked difference in their CaM binding behavior. At a Ca(2+) concentration of 200 microM, CaM bound to both isoforms at a ratio of one CaM molecule per one RYR subunit. At a Ca(2+) concentration of <10 nM, CaM bound primarily to alphaRYR and the binding affinity was significantly lower than that at micromolar level of Ca(2+) concentration. Cloning and sequencing of putative CaM binding sites in alphaRYR and betaRYR suggests that differences in primary structures of the CaM binding sites of each RYR isoform may contribute to the differential CaM binding behavior of alphaRYR and betaRYR.
鸟类骨骼肌兴奋-收缩偶联过程中的Ca(2+)释放受两种兰尼碱受体亚型αRYR和βRYR的控制。另外两种蛋白质，二氢吡啶受体（DHPR）和钙调蛋白（CaM），已被证明在调节RYR通道活性中发挥重要作用。在本研究中，我们测量了火鸡骨骼肌中DHPR和RYR的蛋白质含量，得出DHPR与αRYR的比例为1:1，这表明只有一部分αRYR与DHPR相互作用。使用两种CaM衍生物，即可光活化交联探针[(125)I]-Bz-CaM和代谢标记探针[(35)S]CaM，来研究火鸡骨骼肌中CaM与RYR亚型之间的相互作用。αRYR和βRYR在其CaM结合行为上表现出明显差异。在Ca(2+)浓度为200 microM时，CaM以每一个RYR亚基一个CaM分子的比例与两种亚型结合。在Ca(2+)浓度<10 nM时，CaM主要与αRYR结合，且结合亲和力明显低于微摩尔水平Ca(2+)浓度时的亲和力。对αRYR和βRYR中假定的CaM结合位点进行克隆和测序表明，每种RYR亚型的CaM结合位点一级结构的差异可能导致αRYR和βRYR不同的CaM结合行为。
