S-Adenosyl-L-methionine (SAM) was used to probe the functional effects exerted via the cardiac RyR isoform (RyR2) adenine nucleotide binding site. Single channel experiments revealed that SAM applied to the cytoplasmic face of RyR2 had complex voltage dependent effects on channel gating and conductance. At positive transmembrane holding potentials, SAM caused a striking reduction in channel openings and a reduced channel conductance. In contrast, at negative potentials, SAM promoted a clearly resolved subconductance state. At membrane potentials between −75 and −25 mV, the open probability of the subconductance state was independent of voltage. ATP, but not the non-adenosine-based ryanodine receptor (RyR) activator 4-chloro-m-cresol, interfered with the effects of SAM at both negative and positive potentials. This suggests that ATP and SAM interact with a common binding site. Molecular docking showed SAM bound to the adenine nucleotide binding site and formed a hydrogen bond to Glu4886 in the C-terminal end of the S6 alpha helix. In this configuration, SAM may alter the conformation of the RyR2 ion conduction pathway. This work provides novel insight into potential functional outcomes of ligand binding to the RyR adenine nucleotide binding site.

中文翻译：

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S-腺苷甲硫氨酸通过 ATP 结合位点诱导的极性依赖性门控和 RyR2 的独特亚电导

S-腺苷-L-蛋氨酸 (SAM) 用于探测通过心脏 RyR 亚型 (RyR2) 腺嘌呤核苷酸结合位点发挥的功能效应。单通道实验表明，应用于 RyR2 细胞质表面的 SAM 对通道门控和电导具有复杂的电压依赖性影响。在正跨膜保持电位下，SAM 导致通道开口显着减少和通道电导降低。相反，在负电位下，SAM 促进了明显分辨的亚导状态。在 -75 和 -25 mV 之间的膜电位下，亚导状态的打开概率与电压无关。ATP，但不是基于非腺苷的兰尼碱受体 (RyR) 激活剂 4-氯间甲酚，干扰了 SAM 在负电位和正电位下的作用。这表明 ATP 和 SAM 与一个共同的结合位点相互作用。分子对接显示 SAM 与腺嘌呤核苷酸结合位点结合，并在 S6 α 螺旋的 C 末端与 Glu4886 形成氢键。在这种配置中，SAM 可能会改变 RyR2 离子传导通路的构象。这项工作为配体与 RyR 腺嘌呤核苷酸结合位点结合的潜在功能结果提供了新的见解。