Tian Changlin, Vanoye Carlos G, Kang Congbao, Welch Richard C, Kim Hak Jun, George Alfred L, Sanders Charles R
Department of Biochemistry, Center for Structural Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-8725, USA.
Biochemistry. 2007 Oct 16;46(41):11459-72. doi: 10.1021/bi700705j. Epub 2007 Sep 25.
KCNE1, also known as minK, is a member of the KCNE family of membrane proteins that modulate the function of KCNQ1 and certain other voltage-gated potassium channels (KV). Mutations in human KCNE1 cause congenital deafness and congenital long QT syndrome, an inherited predisposition to potentially life-threatening cardiac arrhythmias. Although its modulation of KCNQ1 function has been extensively characterized, many questions remain regarding KCNE1's structure and location within the channel complex. In this study, KCNE1 was overexpressed in Escherichia coli and purified. Micellar solutions of the protein were then microinjected into Xenopus oocytes expressing KCNQ1 channels, followed by electrophysiological recordings aimed at testing whether recombinant KCNE1 can co-assemble with the channel. Nativelike modulation of channel properties was observed following injection of KCNE1 in lyso-myristoylphosphatidylglycerol (LMPG) micelles, indicating that KCNE1 is not irreversibly misfolded and that LMPG is able to act as a vehicle for delivering membrane proteins into the membranes of viable cells. 1H-15N TROSY NMR experiments indicated that LMPG micelles are well-suited for structural studies of KCNE1, leading to assignment of its backbone resonances and to relaxation studies. The chemical shift data confirmed that KCNE1's secondary structure includes several alpha-helices and demonstrated that its distal C-terminus is disordered. Surprisingly, for KCNE1 in LMPG micelles, there appears to be a break in alpha-helicity at sites 59-61, near the middle of the transmembrane segment, a feature that is accompanied by increased local backbone mobility. Given that this segment overlaps with sites 57-59, which are known to play a critical role in modulating KCNQ1 channel activation kinetics, this unusual structural feature likely has considerable functional relevance.
KCNE1，也被称为minK，是膜蛋白KCNE家族的成员之一，该家族可调节KCNQ1及某些其他电压门控钾通道（KV）的功能。人类KCNE1基因的突变会导致先天性耳聋和先天性长QT综合征，这是一种遗传性疾病，易引发危及生命的心律失常。尽管其对KCNQ1功能的调节已得到广泛研究，但关于KCNE1在通道复合物中的结构和位置仍存在许多问题。在本研究中，KCNE1在大肠杆菌中过表达并纯化。然后将该蛋白的胶束溶液显微注射到表达KCNQ1通道的非洲爪蟾卵母细胞中，随后进行电生理记录，以测试重组KCNE1是否能与该通道共同组装。在注射溶菌 - 肉豆蔻酰磷脂酰甘油（LMPG）胶束中的KCNE1后，观察到通道特性的天然样调节，这表明KCNE1没有发生不可逆的错误折叠，并且LMPG能够作为将膜蛋白递送至活细胞细胞膜的载体。1H - 15N TROSY NMR实验表明，LMPG胶束非常适合用于KCNE1的结构研究，从而实现了其主链共振的归属和弛豫研究。化学位移数据证实KCNE1的二级结构包括几个α螺旋，并表明其远端C末端是无序的。令人惊讶的是，对于LMPG胶束中的KCNE1，在跨膜段中部附近的59 - 61位点似乎存在α螺旋性的中断，这一特征伴随着局部主链流动性的增加。鉴于该片段与已知在调节KCNQ1通道激活动力学中起关键作用的57 - 59位点重叠，这种不寻常的结构特征可能具有相当大的功能相关性。
