Gating kinetics and ligand sensitivity modified by phosphorylation of cardiac ryanodine receptors.
Gating kinetics and ligand sensitivity modified by phosphorylation of cardiac ryanodine receptors.
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通过心脏兰尼碱受体磷酸化改变门控动力学和配体敏感性。
DOI：
10.1007/s00424-002-0791-3
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发表时间：
2002
期刊：
影响因子：
--
通讯作者：
Imanaga,Issei
中科院分区：
文献类型：
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作者：
Uehara,Akira;Yasukochi,Midori;Mejía-Alvarez,Rafael;Fill,Michael;Imanaga,Issei
研究了蛋白激酶A（PKA）依赖性磷酸化对兰尼碱受体（RyR）通道稳态门控动力学的影响。将犬心脏RyR的单通道活性重建成平面脂质双层。外源性PKA增加了单通道开放概率（PO）的本地和纯化的心脏RyR通道，与ATP和Mg 2+预孵育后。PKA对RyR通道的作用仅在ATP和腺苷5′-O-（3-硫代三磷酸）（ATPγS）存在时发生，而在5′-腺苷酰亚胺二磷酸（AMP-PCP）存在时不发生。因此，PKA的作用需要可水解的ATP类似物的存在。PKA诱导的通道激活被特异性PKA抑制剂阻断。所有这些结果都证实RyR通道可以被外源蛋白激酶磷酸化。ATP和Mg ~（2+）作为生理配体显著改变PKA处理前单个RyR通道的门控动力学。相比之下，PKA处理后，ATP和Mg 2+都没有显着改变这些通道的门控动力学。因此，PKA依赖性磷酸化降低了ATP和Mg 2+的表观敏感性，在大多数的门控参数的单一RyR通道。磷酸化的RyR通道打开和关闭更频繁，保持打开时间更长，保持关闭时间更短。获得的停留时间直方图表明，磷酸化和去磷酸化的通道具有显着不同的开放和关闭的动力学在生理细胞质浓度的Mg和ATP。
The effects of protein-kinase- (PKA-) dependent phosphorylation on the stationary gating kinetics of single ryanodine receptor (RyR) channels was defined. The single-channel activity from canine cardiac RyR was reconstituted into planar lipid bilayers. Exogenously applied PKA increased the single-channel open probability (Po) of both native and purified cardiac RyR channels, after preincubation with ATP and Mg2+. The action of PKA on the RyR channel occurred only in the presence of ATP and adenosine 5′-O-(3-thiotriphosphate) (ATPγS), but not in the presence of 5′-adenylimidodiphosphate (AMP-PCP). Thus, the action of PKA requires the presence of a hydrolyzable ATP analog. PKA-induced channel activation was blocked by specific PKA inhibitors. All these results confirmed that the RyR channel can be phosphorylated by exogenous protein kinase. The gating kinetics of single RyR channels before PKA treatment were significantly altered by ATP and Mg2+as physiological ligands. In contrast, after PKA treatment, neither ATP nor Mg2+significantly alters the gating kinetics of these channels. PKA-dependent phosphorylation thus decreases the ATP and Mg2+apparent sensitivity in most of the gating parameters of single RyR channels. The phosphorylated RyR channels open and close more frequently, stay open for longer, and stay closed for shorter periods. The dwell-time histograms obtained demonstrate that the phosphorylated and the dephosphorylated channels have strikingly different open and closed kinetics at physiological cytoplasmic concentrations of Mg and ATP.