A shortcut to a skeletal muscle DHPR knock-in?
A shortcut to a skeletal muscle DHPR knock-in?
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骨骼肌 DHPR 敲入的捷径？
DOI：
10.1113/jphysiol.2011.216234
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发表时间：
2011
期刊：
影响因子：
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通讯作者：
Polster,Alexander
中科院分区：
文献类型：
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作者：
Bannister,RogerA;Polster,Alexander
哺乳动物骨骼肌的兴奋-收缩（EC）偶联依赖于l型Ca2+通道（或1,4 -二氢吡啶受体；DHPR）和1型ryanodine受体（RyR1）之间的分子间通信。DHPR中的构象重排响应于横向管状膜去极化，通过两个通道之间的物理耦合传导到RyR1，这在很大程度上独立于通过DHPR的任何Ca2+进入（见Karunasekara et al. 2009）。除了从DHPR传输到RyR1的“正向”信号外，构象耦合还支持从RyR1到DHPR的“逆行”信号，该信号增加了宏观l型电流振幅（Nakai et al. 1996）。虽然大分子信号复合体的许多其他组分支持这种通信模式（例如triadin， JP-45, Homer, FKBP12，结亲蛋白，DHPR γ和α2δ-1亚基等），但迄今为止已知只有RyR1和DHPR α1S和β1a亚基对EC偶联至关重要。缺乏这三种蛋白质中的任何一种的小鼠死于膈肌麻痹引起的围产期窒息。不用说，这些新生小鼠无法收缩膈肌，阻碍了对成年骨骼肌中EC偶联的基本机制的研究。幸运的是，肌管可以很容易地从胎儿或新生儿幼崽中培养出来，因此，肌管已被证明是研究DHPR和RyR1之间相互作用的非常有用的模型系统。该体外系统使修饰的RyR1， α1S和β1a亚基在真实零背景下表达，从而促进了这些支持DHPR和RyR1之间通信的必需蛋白区域的鉴定和功能表征。尽管使用无肌管研究EC耦合有许多优点，但该系统的明显缺点是在塑料培养皿中生长的肌管不是分化的肌纤维。特别是，由横小管膜和成人肌肉的肌浆网（SR）形成的完全发育的三联结在培养的肌管中是不存在的，后者有较少组织的质膜- SR连接。由于构象耦合严重依赖于连接的超微结构，肌管和成纤维之间DHPR-RyR1相互作用的机制差异可能存在。鉴于这些潜在的差异，需要一种实验系统，在该系统中，修饰的α1S、β1a或RyR1克隆可以在成人纤维中表达和评估，而不受各自内源蛋白的干扰。
Excitation–contraction(EC) coupling in mammalian skeletal muscle relies on intermolecular communication between the L-type Ca2+ channel (or 1, 4-dihydropyridine receptor; DHPR) and the type 1 ryanodine receptor (RyR1). Conformational rearrangements in the DHPR that occur in response to transverse tubular membrane depolarization are transduced to RyR1 via a physical coupling between the two channels that is largely independent of any Ca2+ entry via the DHPR (reviewed in Karunasekara et al. 2009). In addition to this ‘orthograde’signal transmitted from the DHPR to RyR1, conformational coupling also supports a ‘retrograde’signal from RyR1 to the DHPR which increases macroscopic L-type current amplitude (Nakai et al. 1996). Although there are many other components of the macromolecular signalling complex that support this mode of communication (eg triadin, JP-45, Homer, FKBP12, junctophilins, DHPR γ and α2δ-1 subunits, etc.), only RyR1 and the DHPR α1S and β1a subunits are to date known to be essential for EC coupling. Mice null for any one of these three proteins die perinatally from asphyxia resulting from diaphragm paralysis. Needless to say, the inability of these neonatal mice to contract their diaphragms has precluded investigation of the basic mechanism of EC coupling in adult skeletal muscle. Fortunately, myotubes can be easily cultured from fetal or neonatal pups, and for this reason, have proven to be an exceptionally useful model system for the study of the interaction between the DHPR and RyR1. This in vitro system has enabled the expression of modified RyR1, α1S and β1a subunits in true null backgrounds, thereby facilitating identification and functional characterization of regions of these essential proteins that support communication between the DHPR and RyR1.Despite the many advantages of using null myotubes to study EC coupling, the obvious shortcoming of this system is that myotubes growing in a plastic culture dish are not differentiated muscle fibres. In particular, the fully developed triad junctions formed by the membranes of the transverse tubules and the sarcoplasmic reticulum (SR) of adult muscle are absent in cultured myotubes, which have less organized plasma membrane–SR junctions. Since conformational coupling is critically dependent on junctional ultrastructure, mechanistic differences in the DHPR–RyR1 interaction between myotubes and adult fibres may exist. In light of these potential differences, there is a need for an experimental system in which modified α1S, β1a or RyR1 clones can be expressed and evaluated in adult fibres without interference from the respective endogenous proteins.