Centro Interdisciplinario de Neurociencia de Valparaíso, Universidad de Valparaíso, Valparaíso, Chile.
Centro Interdisciplinario de Neurociencia de Valparaíso, Universidad de Valparaíso, Valparaíso, Chile; Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Santiago, Chile.
Biophys J. 2018 Jun 5;114(11):2493-2497. doi: 10.1016/j.bpj.2018.04.008. Epub 2018 Apr 26.
Two families of accessory proteins, β and γ, modulate BK channel gating and pharmacology. Notably, in the absence of internal Ca, the γ1 subunit promotes a large shift of the BK conductance-voltage curve to more negative potentials. However, very little is known about how α- and γ1 subunits interact. In particular, the association stoichiometry between both subunits is unknown. Here, we propose a method to answer this question using lanthanide resonance energy transfer. The method assumes that the kinetics of lanthanide resonance energy transfer-sensitized emission of the donor double-labeled α/γ1 complex is the linear combination of the kinetics of the sensitized emission in single-labeled complexes. We used a lanthanide binding tag engineered either into the α- or the γ1 subunits to bind Tb as the donor. The acceptor (BODIPY) was attached to the BK pore-blocker iberiotoxin. We determined that γ1 associates with the α-subunit with a maximal 1:1 stoichiometry. This method could be applied to determine the stoichiometry of association between proteins within heteromultimeric complexes.
两类辅助蛋白家族，β和γ，调节 BK 通道的门控和药理学特性。值得注意的是，在没有内部 Ca 的情况下，γ1 亚基促进 BK 电导-电压曲线向更负的电位发生大的移动。然而，人们对 α 和 γ1 亚基如何相互作用知之甚少。特别是，两个亚基之间的缔合化学计量比是未知的。在这里，我们提出了一种使用镧系元素共振能量转移来回答这个问题的方法。该方法假设镧系元素共振能量转移敏化供体双标记的 α/γ1 复合物的发射动力学是单标记复合物的敏化发射动力学的线性组合。我们使用镧系元素结合标签工程化到 α 或 γ1 亚基中以结合 Tb 作为供体。受体（BODIPY）被连接到 BK 通道阻断剂 Iberiotoxin 上。我们确定 γ1 与 α-亚基以最大 1:1 的化学计量比结合。该方法可用于确定异源多聚体复合物中蛋白质之间的缔合化学计量比。
