The Contribution of RCK Domains to Human BK Channel Allosteric Activation*
The Contribution of RCK Domains to Human BK Channel Allosteric Activation*
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DOI：
10.1074/jbc.m112.346171
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发表时间：
2012-05
期刊：
影响因子：
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通讯作者：
N. Savalli;A. Pantazis;T. Yusifov;D. Sigg;R. Olcese
中科院分区：
文献类型：
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作者：
N. Savalli;A. Pantazis;T. Yusifov;D. Sigg;R. Olcese
背景：在 BK 通道中，Ca2+ 和电压传感器以变构方式连接到孔。结果：我们光学解决了由 Ca2+ 与细胞内结构域 RCK1 和 RCK2 结合引发的电压传感器重排。损害 RCK2 就消除了这种变构效应。结论：RCK2 Ca2+ 传感器是电压传感器激活变构促进所必需的。意义：RCK1 和 RCK2 Ca2+ 传感器在功能上并不同源。大电导电压和 Ca2+ 激活的 K+ (BK) 通道是细胞过程的有效调节器，包括神经元放电、突触传递、耳蜗毛细胞调节、胰岛素释放和平滑肌张力。其独特的激活途径依赖于结构上不同的调节域，包括一个跨膜电压感应域 (VSD) 和每个亚基两个细胞内高亲和力 Ca2+ 感应位点（位于 RCK1 和 RCK2 域）。四对 RCK1 和 RCK2 结构域形成一个 Ca2+ 感应装置，称为“门环”。电压和 Ca2+ 传感装置之间的变构相互作用是 BK 通道功能的基本机制。使用电压钳荧光测定法和细胞内笼状 Ca2+ 的紫外光解，我们光学解析了 Ca2+ 与门环结合引起的 VSD 激活。细胞内 Ca2+ 浓度 ([Ca2+]i) 的突然增加引起通道开放和 VSD 激活的电压依赖性的超极化转变，由位于 S4 跨膜段上侧的荧光团标记位置 202 报告。位于 RCK2 结构域的 Ca2+ 传感器的中和消除了 [Ca2+]i 增加对 VSD 重排的影响。另一方面，参与 Ca2+ 传感的 RCK1 残基的突变并没有阻止 Ca2+ 释放对 VSD 的影响，揭示了 RCK1 和 RCK2 与 VSD 之间功能上不同的相互作用。统计力学模型量化了两个不同位点的 Ca2+ 关联、电压传感器激活和 BK 通道开放之间复杂的热力学相互作用。
Background: In BK channels, Ca2+ and voltage sensors are allosterically connected to the pore. Results: We optically resolved voltage sensor rearrangements, initiated by Ca2+ binding to the intracellular domains RCK1 and RCK2. Impairing the RCK2 abolished this allosteric effect. Conclusion: The RCK2 Ca2+ sensor is required for the allosteric facilitation of voltage sensor activation. Significance: RCK1 and RCK2 Ca2+ sensors are not functionally homologous. Large conductance voltage- and Ca2+-activated K+ (BK) channels are potent regulators of cellular processes including neuronal firing, synaptic transmission, cochlear hair cell tuning, insulin release, and smooth muscle tone. Their unique activation pathway relies on structurally distinct regulatory domains including one transmembrane voltage-sensing domain (VSD) and two intracellular high affinity Ca2+-sensing sites per subunit (located in the RCK1 and RCK2 domains). Four pairs of RCK1 and RCK2 domains form a Ca2+-sensing apparatus known as the “gating ring.” The allosteric interplay between voltage- and Ca2+-sensing apparati is a fundamental mechanism of BK channel function. Using voltage-clamp fluorometry and UV photolysis of intracellular caged Ca2+, we optically resolved VSD activation prompted by Ca2+ binding to the gating ring. The sudden increase of intracellular Ca2+ concentration ([Ca2+]i) induced a hyperpolarizing shift in the voltage dependence of both channel opening and VSD activation, reported by a fluorophore labeling position 202, located in the upper side of the S4 transmembrane segment. The neutralization of the Ca2+ sensor located in the RCK2 domain abolished the effect of [Ca2+]i increase on the VSD rearrangements. On the other hand, the mutation of RCK1 residues involved in Ca2+ sensing did not prevent the effect of Ca2+ release on the VSD, revealing a functionally distinct interaction between RCK1 and RCK2 and the VSD. A statistical-mechanical model quantifies the complex thermodynamics interplay between Ca2+ association in two distinct sites, voltage sensor activation, and BK channel opening.