Department of Cellular and Molecular Pharmacology, Juntendo University Graduate School of Medicine, Tokyo 113-8421, Japan.
Department of Cellular and Molecular Pharmacology, Juntendo University Graduate School of Medicine, Tokyo 113-8421, Japan.
J Biol Chem. 2011 Oct 14;286(41):35571-35577. doi: 10.1074/jbc.M111.255240. Epub 2011 Aug 23.
The type 1 ryanodine receptor (RyR1) is a Ca(2+) release channel found in the sarcoplasmic reticulum of skeletal muscle and plays a pivotal role in excitation-contraction coupling. The RyR1 channel is activated by a conformational change of the dihydropyridine receptor upon depolarization of the transverse tubule, or by Ca(2+) itself, i.e. Ca(2+)-induced Ca(2+) release (CICR). The molecular events transmitting such signals to the ion gate of the channel are unknown. The S4-S5 linker, a cytosolic loop connecting the S4 and S5 transmembrane segments in six-transmembrane type channels, forms an α-helical structure and mediates signal transmission in a wide variety of channels. To address the role of the S4-S5 linker in RyR1 channel gating, we performed alanine substitution scan of N-terminal half of the putative S4-S5 linker (Thr(4825)-Ser(4829)) that exhibits high helix probability. The mutant RyR1 was expressed in HEK cells, and CICR activity was investigated by caffeine-induced Ca(2+) release, single-channel current recordings, and [(3)H]ryanodine binding. Four mutants (T4825A, I4826A, S4828A, and S4829A) had reduced CICR activity without changing Ca(2+) sensitivity, whereas the L4827A mutant formed a constitutive active channel. T4825I, a disease-associated mutation for malignant hyperthermia, exhibited enhanced CICR activity. An α-helical wheel representation of the N-terminal S4-S5 linker provides a rational explanation to the observed activities of the mutants. These results suggest that N-terminal half of the S4-S5 linker may form an α-helical structure and play an important role in RyR1 channel gating.
1 型兰尼碱受体（RyR1）是骨骼肌肌浆网中的 Ca（2+）释放通道，在兴奋-收缩偶联中起着关键作用。RyR1 通道在横管去极化时通过二氢吡啶受体的构象变化激活，或者通过 Ca（2+）本身，即 Ca（2+）诱导的 Ca（2+）释放（CICR）。将这些信号传递到通道离子门的分子事件尚不清楚。S4-S5 接头是连接六跨膜通道中 S4 和 S5 跨膜片段的胞质环，形成 α-螺旋结构，并在各种通道中介导信号传递。为了研究 S4-S5 接头在 RyR1 通道门控中的作用，我们对具有高螺旋概率的假定 S4-S5 接头（Thr（4825）-Ser（4829））的 N 端进行了丙氨酸取代扫描。突变型 RyR1 在 HEK 细胞中表达，并通过咖啡因诱导的 Ca（2+）释放、单通道电流记录和[（3）H]ryanodine 结合来研究 CICR 活性。四个突变体（T4825A、I4826A、S4828A 和 S4829A）的 CICR 活性降低而不改变 Ca（2+）敏感性，而 L4827A 突变体形成组成型激活通道。T4825I 是恶性高热的一种疾病相关突变，表现出增强的 CICR 活性。N 端 S4-S5 接头的α-螺旋轮表示提供了对观察到的突变体活性的合理解释。这些结果表明，S4-S5 接头的 N 端可能形成α-螺旋结构，并在 RyR1 通道门控中发挥重要作用。
