College of Health & Life Sciences, Aston University, Aston Triangle, Birmingham B4 7ET, U.K.
School of Biosciences, University of Birmingham, Birmingham, U.K.
Biochem J. 2022 Aug 12;479(15):1609-1619. doi: 10.1042/BCJ20210628.
Human BK channels are large voltage and Ca2+-activated K+ channels, involved in several important functions within the body. The core channel is a tetramer of α subunits, and its function is modulated by the presence of β and γ accessory subunits. BK channels composed of α subunits, as well as BK channels composed of α and β1 subunits, were successfully solubilised from HEK cells with styrene maleic acid (SMA) polymer and purified by nickel affinity chromatography. Native SMA-PAGE analysis of the purified proteins showed the α subunits were extracted as a tetramer. In the presence of β1 subunits, they were co-extracted with the α subunits as a heteromeric complex. Purified SMA lipid particles (SMALPs) containing BK channel could be inserted into planar lipid bilayers (PLB) and single channel currents recorded, showing a high conductance (≈260 pS), as expected. The open probability was increased in the presence of co-purified β1 subunits. However, voltage-dependent gating of the channel was restricted. In conclusion, we have demonstrated that SMA can be used to effectively extract and purify large, complex, human ion channels, from low expressing sources. That these large channels can be incorporated into PLB from SMALPs and display voltage-dependent channel activity. However, the SMA appears to reduce the voltage dependent gating of the channels.
人 BK 通道是一种大电压和 Ca2+激活的 K+通道，参与体内的几种重要功能。核心通道是由 α 亚基组成的四聚体，其功能由 β 和 γ 辅助亚基的存在调节。用苯乙烯马来酸（SMA）聚合物从 HEK 细胞中成功溶解并通过镍亲和层析纯化由 α 亚基组成的 BK 通道以及由 α 和 β1 亚基组成的 BK 通道。纯化蛋白的天然 SMA-PAGE 分析表明，α 亚基作为四聚体被提取。在 β1 亚基存在下，它们与 α 亚基一起作为异源复合物被共同提取。含有 BK 通道的纯化 SMA 脂质颗粒（SMALPs）可插入平面脂质双层（PLB）并记录单通道电流，表现出高电导率（≈260 pS），这是预期的。在共纯化的 β1 亚基存在下，开放概率增加。然而，通道的电压依赖性门控受到限制。总之，我们已经证明 SMA 可以有效地从低表达源中提取和纯化大型、复杂的人类离子通道。这些大通道可以从 SMALPs 掺入 PLB 并显示电压依赖性通道活性。然而，SMA 似乎会降低通道的电压依赖性门控。
