Kim Hyunyoung, Jo Sooyeon, Song Hye-Jin, Park Zee-Yong, Park Chul-Seung
Department Life Science, Gwangju Institute of Science and Technology (GIST), Gwangju, Korea.
Proteomics. 2007 Aug;7(15):2591-602. doi: 10.1002/pmic.200700185.
The activity and localization of large-conductance Ca2+ -activated K+ (BKCa) channels are known to be modulated by several different proteins. Although many binding partners have been identified via yeast two-hybrid screening, this method may not detect certain classes of interacting proteins such as low affinity binding proteins or multi-component protein complexes. In this study, we employed mass spectrometry to identify proteins that interact with BKCa channels. We expressed and purified the 'tail domain' of the rat BKCa channel alpha-subunit, a 54-kDa region that is crucial for expression and functional activity of the channel. Using rat brain lysate and purified 'tail domain', we identified several novel proteins that interact with the BKCa channel. These included the myelin basic protein (MBP), upon which we performed subsequent biochemical and electrophysiological studies. Interaction between the BKCa channel and MBP was confirmed in vivo and in vitro. MBP co-expression affected the Ca2+ -dependent activation of the BKCa channel by increasing its Ca2+ sensitivity. Moreover, we showed that calmodulin (CaM) interacts with the BKCa channel via MBP. Since CaM is a key regulator of many Ca2+ -dependent processes, it may be recruited by MBP to the vicinity of the BKCa channel, modulating its functional activity.
已知大电导钙激活钾（BKCa）通道的活性和定位受几种不同蛋白质的调节。尽管通过酵母双杂交筛选已鉴定出许多结合伴侣，但该方法可能无法检测到某些类型的相互作用蛋白，如低亲和力结合蛋白或多组分蛋白复合物。在本研究中，我们采用质谱法鉴定与BKCa通道相互作用的蛋白质。我们表达并纯化了大鼠BKCa通道α亚基的“尾域”，这是一个对通道的表达和功能活性至关重要的54 kDa区域。利用大鼠脑裂解物和纯化的“尾域”，我们鉴定出了几种与BKCa通道相互作用的新蛋白质。其中包括髓鞘碱性蛋白（MBP），我们随后对其进行了生化和电生理研究。在体内和体外均证实了BKCa通道与MBP之间的相互作用。MBP共表达通过增加其对Ca2+的敏感性影响BKCa通道的Ca2+依赖性激活。此外，我们表明钙调蛋白（CaM）通过MBP与BKCa通道相互作用。由于CaM是许多Ca2+依赖性过程的关键调节因子，它可能被MBP招募到BKCa通道附近，调节其功能活性。
