a Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology , Huazhong University of Science and Technology , Wuhan , China.
b State Key Laboratory of Bio-organic and Natural Product Chemistry , Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences , Shanghai , China.
Channels (Austin). 2019 Dec;13(1):17-32. doi: 10.1080/19336950.2018.1551660.
Large-conductance Ca-activated K (BK) channels are composed of a pore-forming α and a variable number of auxiliary β subunits and play important roles in regulating excitability, action potential waveforms and firing patterns, particularly in neurons and endocrine and cardiovascular cells. The β2 subunits increase the diversity of gating and pharmacological properties. Its extracellular loop contains eight cysteine residues, which can pair to form a high-order structure, underlying the stability of the extracellular loop of β2 subunits and the functional effects on BK channels. However, how these cysteines form disulfide bonds still remains unclear. To address this, based on the fact that the rectification and association of BK α to β2 subunits are highly sensitive to disruption of the disulfide bonds in the extracellular loop of β2, we developed a rectification ratio based assay by combining the site-directed mutagenesis, electrophysiology and enzymatic cleavage. Three disulfide bonds: C1(C84)-C5(C113), C3(C101)-C7(C148) and C6(C142)-C8C(174) are successfully deduced in β2 subunit in complex with a BK α subunit, which are helpful to predict structural model of β2 subunits through computational simulation and to understand the interface between the extracellular domain of the β subunits and the pore-forming α subunit.
大电导钙激活钾（BK）通道由一个孔形成的α亚基和一个可变数量的辅助β亚基组成，在调节兴奋性、动作电位波形和放电模式方面发挥着重要作用，特别是在神经元和内分泌和心血管细胞中。β2 亚基增加了门控和药理学特性的多样性。其细胞外环包含八个半胱氨酸残基，这些残基可以配对形成一个高级结构，这是 β2 亚基细胞外环稳定性和对 BK 通道的功能影响的基础。然而，这些半胱氨酸如何形成二硫键仍然不清楚。为了解决这个问题，基于 BK α 与 β2 亚基的整流和关联对 β2 亚基细胞外环中二硫键的破坏高度敏感的事实，我们开发了一种基于整流比的测定法，该方法结合了定点突变、电生理学和酶切。成功推断出与 BK α 亚基复合的 β2 亚基中的三个二硫键：C1(C84)-C5(C113)、C3(C101)-C7(C148)和 C6(C142)-C8C(174)，这有助于通过计算模拟预测 β2 亚基的结构模型，并了解β 亚基细胞外环与孔形成 α 亚基之间的界面。
