Ryanodine receptor/calcium release channel conformations as reflected in the different effects of propranolol on its ryanodine binding and channel activity.
Ryanodine receptor/calcium release channel conformations as reflected in the different effects of propranolol on its ryanodine binding and channel activity.
复制标题
兰尼定受体/钙释放通道构象反映在普萘洛尔对其兰尼定结合和通道活性的不同影响中。
作者：
Sigalit Zchut;Wei Feng;V. Shoshan
1.普萘洛尔，β-受体阻滞剂，抑制或刺激结合的膜结合和纯化的兰尼碱受体（RyR）取决于测定条件的兰尼碱。在高NaCl浓度下，普萘洛尔增加了ryanodine结合位点（Bmax）的数量，但对结合亲和力没有影响。在0.2 M NaCl的存在下，兰尼碱结合被普萘洛尔抑制。在1.2 mM下获得半最大抑制，在2 mM普萘洛尔下获得完全抑制。在低NaCl浓度下获得的普萘洛尔的抑制作用不能通过将NaCl浓度增加到1 M而恢复。2.已知改变其构象状态的RyR调节剂，如腺嘌呤核苷酸、Ca 2+浓度和pH，改变了普萘洛尔对ryanodine结合的影响。在普萘洛尔存在下和低NaCl浓度下，兰尼碱的结合受到抑制，并且没有表现出Ca（2+）、pH或时间依赖性。3.普萘洛尔立即完全阻断了重组成平面脂质双层的RyR通道开放。心得安修饰的非活性通道被ATP重新激活到亚传导状态（约为对照电导的40%）。4.利多卡因（一种刺激性药物）或丁卡因（一种抑制性药物）和普萘洛尔在0.2或1.0 M NaCl，分别之间的竞争实验表明，存在不同的相互作用的局部麻醉剂和普萘洛尔网站。5.这些结果表明，普萘洛尔直接与RyR相互作用，并修改其ryanodine结合和单通道活动。普萘洛尔的影响被改变的RyR构象状态，这表明其可能用作RyR的构象探针。
1. Propranolol, a beta-blocker, inhibited or stimulated ryanodine binding to both the membrane-bound and purified ryanodine receptor (RyR) depending on the assay conditions. At high NaCl concentrations, propranolol increased the number of ryanodine-binding sites (Bmax) with no effect on the binding affinity. In the presence of 0.2 M NaCl, ryanodine binding was inhibited by propranolol. Half-maximal inhibition was obtained at 1.2 mM and complete inhibition at 2 mM propranolol. The inhibitory effect of propranolol obtained at low NaCl concentration was not restored by increasing the NaCl concentration to 1 M. 2. Modulators of the RyR that are known to alter its conformational states, such as adenine nucleotides, Ca2+ concentration and pH, modified the effect of propranolol on ryanodine binding. In the presence of propranolol and at low NaCl concentrations, ryanodine binding was inhibited and showed no Ca(2+)-, pH-, or time-dependence. 3. Propranolol immediately and completely blocked the channel opening of RyR reconstituted into a planar lipid bilayer. Propranolol-modified non-active channel was reactivated to a subconductive state (about 40% of the control conductance) by ATP. 4. Competition experiments between lidocaine (a stimulatory drug) or tetracaine (an inhibitory drug) and propranolol at 0.2 or 1.0 M NaCl, respectively, suggest the existence of different interaction sites for local anaesthetics and propranolol. 5. These results suggest that propranolol interacts directly with the RyR and modifies its ryanodine binding and single-channel activities. Propranolol effects are altered by the RyR conformational state, suggesting its possible use as a conformational probe for RyR.