Accessibility of targeted DHPR sites to streptavidin and functional effects of binding on EC coupling.
Accessibility of targeted DHPR sites to streptavidin and functional effects of binding on EC coupling.
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目标 DHPR 位点对链霉亲和素的可及性以及结合对 EC 偶联的功能影响。
DOI：
10.1085/jgp.200609730
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发表时间：
2007
期刊：
影响因子：
--
通讯作者：
Beam,KurtG
中科院分区：
文献类型：
--
作者：
Lorenzon,NancyM;Beam,KurtG
在骨骼肌中，质膜（PM）中的二氢吡啶受体（DHPR）作为Ca2+通道和兴奋-收缩（EC耦合）的电压传感器，通过肌浆网（SR）膜中的1型ryanodine受体（RyR1）触发Ca2+释放。除了功能连接外，这两种蛋白质在结构上也相互连接，但这些连接的身份仍然未知。为了解决这一问题，我们在肌管中表达了DHPR α1或β1亚基，并将生物素受体结构域融合到目标位点，以表达相应的内源性DHPR亚基。经过皂素渗透后，~ 60 kd链亲和素分子可以进入β1aN和C端，α1SN端和近端II-III环（残基671-686）。Steptavidin在注入活体肌管后也能进入这些部位。然而，在皂素渗透的肌管中，α1SC末端的位点要么是不可接近的，要么是有条件可接近的，这表明这些c -末端区域可能存在于PM/SR连接中被其他蛋白质（如RyR1）阻断的构象中。注射链霉亲和素与β1aN或C端或α1SN端结合对电诱发收缩没有影响。相比之下，链霉亲和素与近端α1SII-III环结合可消除这种收缩，而不影响激动剂诱导的通过RyR1释放Ca2+。此外，EC偶联的阻断似乎不是由DHPR的整体扭曲引起的，这支持了α 1si - iii环的构象改变是骨骼肌EC偶联所必需的假设。
In skeletal muscle, the dihydropyridine receptor (DHPR) in the plasma membrane (PM) serves as a Ca2+channel and as the voltage sensor for excitation–contraction (EC coupling), triggering Ca2+release via the type 1 ryanodine receptor (RyR1) in the sarcoplasmic reticulum (SR) membrane. In addition to being functionally linked, these two proteins are also structurally linked to one another, but the identity of these links remains unknown. As an approach to address this issue, we have expressed DHPR α1Sor β1asubunits, with a biotin acceptor domain fused to targeted sites, in myotubes null for the corresponding, endogenous DHPR subunit. After saponin permeabilization, the ∼60-kD streptavidin molecule had access to the β1aN and C termini and to the α1SN terminus and proximal II–III loop (residues 671–686). Steptavidin also had access to these sites after injection into living myotubes. However, sites of the α1SC terminus were either inaccessible or conditionally accessible in saponin- permeabilized myotubes, suggesting that these C-terminal regions may exist in conformations that are occluded by other proteins in PM/SR junction (e.g., RyR1). The binding of injected streptavidin to the β1aN or C terminus, or to the α1SN terminus, had no effect on electrically evoked contractions. By contrast, binding of streptavidin to the proximal α1SII–III loop abolished such contractions, without affecting agonist-induced Ca2+release via RyR1. Moreover, the block of EC coupling did not appear to result from global distortion of the DHPR and supports the hypothesis that conformational changes of the α1SII–III loop are necessary for EC coupling in skeletal muscle.
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DOI：
10.1073/pnas.88.23.10778
发表时间：
1991-12-01
影响因子：
11.1
作者：
DEJONGH, KS;WARNER, C;CATTERALL, WA
通讯作者：
CATTERALL, WA
影响因子：
4.8
作者：
Nakai, J;Tanabe, T;Beam, KG
通讯作者：
Beam, KG
RYR1 和 L 型钙通道通过钙调蛋白结合域偶联。
影响因子：
4.8
作者：
S. Sencer;R. Papineni;D. B. Halling;P. Pate;J. Krol;J. Z. Zhang;S. Hamilton
通讯作者：
S. Hamilton
RYR1和RYR3在骨骼肌钙释放单位的组装中具有不同的作用。
影响因子：
3.4
作者：
F. Protasi;H. Takekura;Y. Wang;S. Chen;G. Meissner;P. Allen;C. Franzini
通讯作者：
C. Franzini
DOI：
10.1083/jcb.107.6.2587
发表时间：
1988-12
期刊：
The Journal of cell biology
影响因子：
--
作者：
Block BA;Imagawa T;Campbell KP;Franzini-Armstrong C
通讯作者：
Franzini-Armstrong C