Uehara Akira, Yasukochi Midori, Mejía-Alvarez Rafael, Fill Michael, Imanaga Issei
Department of Physiology, School of Medicine, Fukuoka University, Jonan-ku, 814-0180, Japan.
Pflugers Arch. 2002 May;444(1-2):202-12. doi: 10.1007/s00424-002-0791-3. Epub 2002 Feb 14.
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 ( P(o)) 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) (ATPgammaS), 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.
确定了蛋白激酶A（PKA）依赖性磷酸化对单个兰尼碱受体（RyR）通道静态门控动力学的影响。将犬心脏RyR的单通道活性重组到平面脂质双分子层中。在与ATP和Mg2+预孵育后，外源性应用的PKA增加了天然和纯化的心脏RyR通道的单通道开放概率（P(o)）。PKA对RyR通道的作用仅在存在ATP和腺苷5'-O-(3-硫代三磷酸)（ATPγS）时发生，而在存在5'-腺苷亚氨二磷酸（AMP-PCP）时不发生。因此，PKA的作用需要存在可水解的ATP类似物。PKA诱导的通道激活被特异性PKA抑制剂阻断。所有这些结果证实了RyR通道可被外源性蛋白激酶磷酸化。作为生理配体的ATP和Mg2+显著改变了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Mg2+均未显著改变这些通道的门控动力学。因此，PKA依赖性磷酸化降低了单个RyR通道大多数门控参数中ATP和Mg2+的表观敏感性。磷酸化的RyR通道打开和关闭更频繁，开放时间更长，关闭时间更短。获得的驻留时间直方图表明，在生理细胞质浓度的Mg和ATP下，磷酸化和去磷酸化的通道具有明显不同的开放和关闭动力学。
