Guo W, Jorgensen A O, Jones L R, Campbell K P
Howard Hughes Medical Institute, University of Iowa College of Medicine, Iowa City 52242, USA.
J Biol Chem. 1996 Jan 5;271(1):458-65. doi: 10.1074/jbc.271.1.458.
Triadin is an intrinsic membrane protein first identified in the skeletal muscle junctional sarcoplasmic reticulum and is considered to play an important role in excitation-contraction coupling. Using polyclonal antibodies to skeletal muscle triadin, we have identified and characterized three isoforms in rabbit cardiac muscle. The cDNAs encoding these three isoforms of triadin have been isolated by reverse transcription-polymerase chain reaction and cDNA library screening. The deduced amino acid sequences show that these proteins are identical in their N-terminal sequences, whereas the C-terminal sequences are distinct from each other and from that of skeletal muscle triadin. Based upon both the amino acid sequences and biochemical analysis, all three triadin isoforms share similar membrane topology with skeletal muscle triadin. Immunofluorescence staining of rabbit cardiac muscle with antibodies purified from the homologous region of triadin shows that cardiac triadin is primarily confined to the I-band region of cardiac myocytes, where the junctional and corbular sarcoplasmic reticulum is located. Furthermore, we demonstrate that the conserved region of the luminal domain of triadin is able to bind both the ryanodine receptor and calsequestrin in cardiac muscle. These results suggest that triadin colocalizes with and binds to the ryanodine receptor and calsequestrin and carries out a function in the lumen of the junctional sarcoplasmic reticulum that is important for both skeletal and cardiac muscle excitation-contraction coupling.
三联蛋白是一种内在膜蛋白，最初在骨骼肌连接肌质网中被鉴定出来，被认为在兴奋 - 收缩偶联中起重要作用。利用针对骨骼肌三联蛋白的多克隆抗体，我们在兔心肌中鉴定并表征了三种同工型。通过逆转录 - 聚合酶链反应和cDNA文库筛选，分离出了编码这三种三联蛋白同工型的cDNA。推导的氨基酸序列表明，这些蛋白质的N端序列相同，而C端序列彼此不同，也与骨骼肌三联蛋白的C端序列不同。基于氨基酸序列和生化分析，所有三种三联蛋白同工型与骨骼肌三联蛋白具有相似的膜拓扑结构。用从三联蛋白同源区域纯化的抗体对兔心肌进行免疫荧光染色表明，心肌三联蛋白主要局限于心肌细胞的I带区域，即连接肌质网和小囊状肌质网所在的区域。此外，我们证明三联蛋白腔结构域的保守区域能够结合心肌中的兰尼碱受体和肌集钙蛋白。这些结果表明，三联蛋白与兰尼碱受体和肌集钙蛋白共定位并结合，并在连接肌质网腔中发挥作用，这对骨骼肌和心肌的兴奋 - 收缩偶联都很重要。
