Divergent functional properties of ryanodine receptor types 1 and 3 expressed in a myogenic cell line
Divergent functional properties of ryanodine receptor types 1 and 3 expressed in a myogenic cell line
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DOI：
10.1016/s0006-3495(00)76492-7
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发表时间：
2000-11-01
影响因子：
3.4
通讯作者：
Pessah, IN
中科院分区：
文献类型：
--
作者：
Fessenden, JD;Wang, YM;Pessah, IN
在肌肉中表达的三种已知的Ryanodine受体(RyR)亚型中，RyR1和RyR2在收缩中具有明确的作用。然而，哺乳动物RyR3的研究一直很困难，因为相对于RyR1或RyR2，RyR3的表达水平较低。利用单纯疱疹病毒1型(HSV-1)无辅助子扩增系统，我们在1B5缺失RyR的肌管中表达了RyR1或RyR3。Western印迹分析表明，转导RyR1或RyR3的细胞表达了正确分子质量的适当RyR亚型。尽管RyR1通道在双层脂膜中表现出预期的脑脊液单一电导，但88个RyR3通道中有74个通道表现出显著的亚电导行为。用FKBP12/12.6选择性抗体进行Western印迹分析表明，RyR1和RyR3表现出的门控行为的差异可能部分是由于RyR3对FKBP12的亲和力较低所致。在钙成像研究中，RyR1恢复了骨骼类型的兴奋-收缩偶联，而RyR3则没有。尽管表达RyR3的肌管对咖啡因的敏感性高于表达RyR1的肌管，但它们对4-氯-间甲酚(CMC)的敏感性要低得多。在表达RyR1的细胞中，观察到咖啡因和CMC反应的再生性钙振荡，但在表达RyR3的1B5细胞中从未见过。在[H-3]ryanodine结合研究中，只有RyR1对CMC敏感，但两种RyR亚型对咖啡因都有反应。在哺乳动物肌肉中表达的RyR1和RyR3之间的这些功能差异可能反映了与辅助蛋白，特别是FKBP12相关的差异，以及调节剂结合位点的结构差异。
Of the three known ryanodine receptor (RyR) isoforms expressed in muscle, RyR1 and RyR2 have well-defined roles in contraction. However, studies on mammalian RyR3 have been difficult because of low expression levels relative to RyR1 or RyR2. Using the herpes simplex virus 1 (HSV-1) helper-free amplicon system, we expressed either RyR1 or RyR3 in 1B5 RyR-deficient myotubes. Western blot analysis revealed that RyR1 - or RyR3-transduced cells expressed the appropriate RyR isoform of the correct molecular mass. Although RyR1 channels exhibited the expected unitary conductance for Csf in bilayer lipid membranes, 74 of 88 RyR3 channels exhibited pronounced subconductance behavior. Western blot analysis with an FKBP12/12.6-selective antibody reveals that differences in gating behavior exhibited by RyR1 and RyR3 may be, in part, the result of lower affinity of RyR3 for FKBP12. In calcium imaging studies, RyR1 restored skeletal-type excitation-contraction coupling, whereas RyR3 did not. Although RyR3-expressing myotubes were more sensitive to caffeine than those expressing RyR1,they were much less sensitive to 4-chloro-m-cresol (CMC). In RyR1-expressing cells, regenerative calcium oscillations were observed in response to caffeine and CMC but were never seen in RyR3-expressing 1B5 cells. In [H-3]ryanodine binding studies, only RyR1 exhibited sensitivity to CMC, but both RyR isoforms responded to caffeine. These functional differences between RyR1 and RyR3 expressed in a mammalian muscle context may reflect differences in association with accessory proteins, especially FKBP12, as well as structural differences in modulator binding sites.