Preparation, Functional Characterization, and NMR Studies of Human KCNE1, a Voltage-Gated Potassium Channel Accessory Subunit Associated with Deafness and Long QT Syndrome

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• 作者：Changlin Tian ; Carlos G. Vanoye ; Congbao Kang ; Richard C. Welch ; Hak Jun Kim ; Alfred L. George ; Jr. ; Charles R. Sanders
• 刊名：Biochemistry
• 出版年：2007
• 出版时间：October 16, 2007
• 年：2007
• 卷：46
• 期：41
• 页码：11459 - 11472
• 全文大小：541K
• 年卷期：v.46,no.41(October 16, 2007)
• ISSN：1520-4995

文摘

KCNE1, also known as minK, is a member of the KCNE family of membrane proteins thatmodulate the function of KCNQ1 and certain other voltage-gated potassium channels (K_V). Mutations inhuman KCNE1 cause congenital deafness and congenital long QT syndrome, an inherited predispositionto potentially life-threatening cardiac arrhythmias. Although its modulation of KCNQ1 function has beenextensively characterized, many questions remain regarding KCNE1's structure and location within thechannel complex. In this study, KCNE1 was overexpressed in Escherichia coli and purified. Micellarsolutions of the protein were then microinjected into Xenopus oocytes expressing KCNQ1 channels,followed by electrophysiological recordings aimed at testing whether recombinant KCNE1 can co-assemblewith the channel. Nativelike modulation of channel properties was observed following injection of KCNE1in lyso-myristoylphosphatidylglycerol (LMPG) micelles, indicating that KCNE1 is not irreversibly misfoldedand that LMPG is able to act as a vehicle for delivering membrane proteins into the membranes of viablecells. ¹H-¹⁵N TROSY NMR experiments indicated that LMPG micelles are well-suited for structuralstudies of KCNE1, leading to assignment of its backbone resonances and to relaxation studies. The chemicalshift data confirmed that KCNE1's secondary structure includes several -helices and demonstrated thatits distal C-terminus is disordered. Surprisingly, for KCNE1 in LMPG micelles, there appears to be abreak in -helicity at sites 59-61, near the middle of the transmembrane segment, a feature that isaccompanied 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 unusualstructural feature likely has considerable functional relevance.