School of Physiology and Pharmacology and Centre for Nanoscience and Quantum Information, University of Bristol, Bristol BS81TD, UK.
J Membr Biol. 2011 Mar;240(1):21-33. doi: 10.1007/s00232-011-9339-9. Epub 2011 Jan 28.
Phosphorylation of the cardiac ryanodine receptor (RyR2) is thought to be important not only for normal cardiac excitation-contraction coupling but also in exacerbating abnormalities in Ca²+ homeostasis in heart failure. Linking phosphorylation to specific changes in the single-channel function of RyR2 has proved very difficult, yielding much controversy within the field. We therefore investigated the mechanistic changes that take place at the single-channel level after phosphorylating RyR2 and, in particular, the idea that PKA-dependent phosphorylation increases RyR2 sensitivity to cytosolic Ca²+. We show that hyperphosphorylation by exogenous PKA increases open probability (P(o)) but, crucially, RyR2 becomes uncoupled from the influence of cytosolic Ca²+; lowering [Ca²+] to subactivating levels no longer closes the channels. Phosphatase (PP1) treatment reverses these gating changes, returning the channels to a Ca²+-sensitive mode of gating. We additionally found that cytosolic incubation with Mg²+/ATP in the absence of exogenously added kinase could phosphorylate RyR2 in approximately 50% of channels, thereby indicating that an endogenous kinase incorporates into the bilayer together with RyR2. Channels activated by the endogenous kinase exhibited identical changes in gating behavior to those activated by exogenous PKA, including uncoupling from the influence of cytosolic Ca²+. We show that the endogenous kinase is both Ca²+-dependent and sensitive to inhibitors of PKC. Moreover, the Ca²+-dependent, endogenous kinase-induced changes in RyR2 gating do not appear to be related to phosphorylation of serine-2809. Further work is required to investigate the identity and physiological role of this Ca²+-dependent endogenous kinase that can uncouple RyR2 gating from direct cytosolic Ca²+ regulation.
肌浆网ryanodine 受体（RyR2）的磷酸化不仅对正常的心脏兴奋-收缩偶联很重要，而且在心力衰竭时加剧 Ca²+稳态的异常中也很重要。将磷酸化与 RyR2 的单通道功能的特定变化联系起来被证明非常困难，这在该领域引起了很大的争议。因此，我们研究了磷酸化 RyR2 后在单通道水平上发生的机制变化，特别是 PKA 依赖性磷酸化增加 RyR2 对细胞质 Ca²+敏感性的想法。我们表明，外源性 PKA 的过度磷酸化增加了开放概率（P(o)），但至关重要的是，RyR2 与细胞质 Ca²+的影响解偶联；将 [Ca²+] 降低到亚激活水平不再关闭通道。磷酸酶（PP1）处理逆转了这些门控变化，使通道恢复到 Ca²+敏感的门控模式。我们还发现，在没有外加激酶的情况下，细胞质中 Mg²+/ATP 的孵育可以使大约 50%的通道中的 RyR2 发生磷酸化，从而表明一种内源性激酶与 RyR2 一起整合到双层中。由内源性激酶激活的通道表现出与由外源性 PKA 激活的通道相同的门控行为变化，包括与细胞质 Ca²+的影响解偶联。我们表明，内源性激酶既依赖 Ca²+，又对 PKC 抑制剂敏感。此外，内源性激酶诱导的 RyR2 门控的 Ca²+-依赖性变化似乎与丝氨酸-2809 的磷酸化无关。需要进一步的工作来研究这种 Ca²+依赖性内源性激酶的身份和生理作用，这种激酶可以使 RyR2 的门控与直接细胞质 Ca²+调节解偶联。
