The function of BK channels extracted and purified within SMALPs.
The function of BK channels extracted and purified within SMALPs.
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SMALP 内提取和纯化的 BK 通道的功能。
DOI：
10.1042/bcj20210628
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发表时间：
2022-08-12
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人类BK通道是大电压和Ca 2+激活的K+通道，参与体内的几种重要功能。核心通道是α亚基的四聚体，其功能受β和γ辅助亚基的存在调节。用苯乙烯马来酸（SMA）聚合物从HEK细胞中成功地溶解了由α亚基组成的BK通道，以及由α和β1亚基组成的BK通道，并通过镍亲和层析纯化。纯化蛋白的非变性SMA-PAGE分析显示α亚基以四聚体形式提取。在β1亚基存在的情况下，它们与α亚基作为异聚复合物共提取。含有BK通道的纯化SMA脂质颗粒（SMALPs）可插入平面脂质双层（PLB）中，并记录到单通道电流，如预期的那样，显示出高电导（约260 pS）。 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可以用于从低表达来源有效地提取和纯化大的、复杂的人离子通道。这些大通道可以从SMALPs整合到PLB中，并显示电压依赖性通道活性。然而，SMA似乎降低了通道的电压依赖性门控。
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.