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.

中文翻译：

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SMALPs中提取纯化BK通道的功能

人体 BK 通道是大电压和 Ca2+ 激活的 K+ 通道，参与体内的几个重要功能。核心通道是 α 亚基的四聚体，其功能受 β 和 γ 辅助亚基的调节。由 α 亚基组成的 BK 通道以及由 α 和 β1 亚基组成的 BK 通道用苯乙烯马来酸 (SMA) 聚合物从 HEK 细胞中成功溶解，并通过镍亲和层析纯化。纯化蛋白质的天然 SMA-PAGE 分析显示 α 亚基被提取为四聚体。在存在 β1 亚基的情况下，它们与 α 亚基作为杂聚复合物共同提取。含有 BK 通道的纯化 SMA 脂质颗粒 (SMALP) 可插入平面脂质双层 (PLB) 并记录单通道电流，显示出高电导 (≈260 pS)，正如预期的那样。在共纯化的 β1 亚基存在下，开放概率增加。然而，通道的电压依赖门控受到限制。总之，我们已经证明 SMA 可用于从低表达来源有效地提取和纯化大型、复杂的人体离子通道。这些大通道可以从 SMALP 整合到 PLB 中，并显示电压依赖性通道活动。然而，SMA 似乎降低了通道的电压相关门控。这些大通道可以从 SMALP 整合到 PLB 中，并显示电压依赖性通道活动。然而，SMA 似乎降低了通道的电压相关门控。这些大通道可以从 SMALP 整合到 PLB 中，并显示电压依赖性通道活动。然而，SMA 似乎降低了通道的电压相关门控。