Dias José M, Vogel Pia D
Department of Biological Sciences, Southern Methodist University, 6501 Airline Rd, Dallas, TX 75275, USA.
Protein J. 2009 Jun;28(5):240-6. doi: 10.1007/s10930-009-9189-9.
Calcium release for muscle contraction in skeletal muscle is mediated in part by the ryanodine receptor 1, RyR1, Ca2+-channel and is strongly affected by intrinsic modulators like Ca2+, Mg2+ and ATP. We showed differential effects on ATP binding in the presence of Ca2+ or Mg2+ ions using ESR spectroscopy and a spin-labeled ATP analog, SL-ATP (Dias et al. Biochemistry 45: 9408-9415, 2006). We here report the effects of RyR1 modulators like ryanodine, caffeine and dantrolene on the ATP binding of RyR1 using the same technique. We present evidence that the exogenous effectors induce changes within RyR1 that lead to different ATP binding characteristics: In the presence of the activating modulator, caffeine, or in the presence of ryanodine, which causes a half-open state of the channel, binding of eight ATP per RyR1 was observed, even in the presence of inhibitory Ca2+, suggestive of a stable "open" channel conformation. In the presence of the inhibitory modulator dantrolene, ATP binding affinity decreased in the presence of activating Ca2+, while in the presence of inhibitory Ca2+, ATP binding affinity increased, but at the same time the number of accessible sites decreased to four, suggestive of a closed conformation of the channel. The results imply that modulation of ATP binding to RyR1 as well as the overall number of accessible ATP binding sites on the channel are crucial for regulation and are in direct correlation with the modified activity of the channel induced by pharmacological agents.
骨骼肌中肌肉收缩的钙释放部分由兰尼碱受体1（RyR1）钙通道介导，并受到Ca2+、Mg2+和ATP等内在调节剂的强烈影响。我们使用电子顺磁共振光谱和自旋标记的ATP类似物SL-ATP，展示了在Ca2+或Mg2+离子存在下对ATP结合的不同影响（迪亚斯等人，《生物化学》45: 9408 - 9415，2006年）。我们在此报告使用相同技术，兰尼碱、咖啡因和丹曲林等RyR1调节剂对RyR1的ATP结合的影响。我们提供的证据表明，外源性效应物会在RyR1内诱导变化，从而导致不同的ATP结合特性：在激活调节剂咖啡因存在下，或在导致通道半开放状态的兰尼碱存在下，即使在抑制性Ca2+存在的情况下，每个RyR1也观察到八个ATP的结合，这表明通道具有稳定的“开放”构象。在抑制性调节剂丹曲林存在下，在激活Ca2+存在时ATP结合亲和力降低，而在抑制性Ca2+存在时，ATP结合亲和力增加，但同时可及位点的数量减少到四个，这表明通道处于关闭构象。结果表明，ATP与RyR1结合的调节以及通道上可及ATP结合位点的总数对于调节至关重要，并且与药物诱导的通道活性改变直接相关。
