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

Abstract

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 (K V ). 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 E. coli and purified. Micellar solutions of the protein were then microinjected into Xenopus oocytes expressing KCNQ1 channels, followed by electrophysiological recordings to test whether recombinant KCNE1 can co-assemble with the channel. Native-like modulation of channel properties was observed following injection of KCNE1 in lysomyristoylphosphatidylglycerol (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. 1 H, 15 N-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 α-helices and demonstrated that its distal Cterminus is disordered. Surprisingly, for KCNE1 in LMPG micelles there appears to be a break in α-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 is likely of considerable functional relevance.Voltage-gated potassium channels (K V ) play a variety of important roles in human health and disease. For example, human KCNQ1 is essential to the cardiac action potential that mediates heartbeat and is also critical for potassium ion homeostasis in the inner ear(1;2). The function of several K V channels is modulated by accessory proteins including K V channel β subunits (Kvβ)(3-6), potassium channel interacting proteins (KCHiP)(7;8), and the KCNE family of † This study was supported by NIH grant R01 DC007416 to CRS, NIH grant HL077188 to ALG, a Vanderbilt School of Medicine Discovery Grant to CGV, and by a postdoctoral fellowship from the American Heart Association to CT (0625586B). Some data were collected in the SEC NMR facility at the University of Georgia, which is supported by US NIH grant P41 GM066340. The NMR assignments for KCNE1 that are reported in this paper have been deposited in the BMRB with access number 15102. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript single transmembrane proteins including KCNE1 and minK-related peptides (MiRPs) (9)(10)(11)(12)(13)(14). KCNE1, also known as minK, co-assembles with KCNQ1 in heart muscle cells...

Biosynthesis and NMR-studies of a double transmembrane domain from the Y4 receptor, a human GPCR

“…A large body of literature supports the basic assumption of the model: For example, proteolysis of membrane proteins resulted in fragments containing entire TM sequences (Huang et al 1981), and chemically or recombinantly synthesized TM peptides spontaneously assembled thereby rescuing receptor activity (Kahn and Engelman 1992;Ridge et al 1995;Martin et al 1999;Wrubel et al 1994). Finally, peptides corresponding to the N and C terminus (Harmar 2001;O'Hara et al 1993), loop domains (Bennett et al 2004;Katragadda et al 2001a, b;Yeagle et al 2000) and transmembrane domains (Katragadda et al 2001a, b;Cohen et al 2008;Zheng et al 2006;Musial-Siwek et al 2008;Tian et al 2007;Lau et al 2008;Mobley et al 2007;Neumoin et al 2007) from GPCRs have been found to fold to distinct secondary structures which in certain cases resembled the structures of the corresponding regions of the intact receptor.…”

Dynamic Partnership between KCNQ1 and KCNE1 and Influence on Cardiac IKs Current Amplitude by KCNE2

“…Vesicle Injection and Oocyte Expression-The procedures were modified from those described previously (38,39). COS-7 cells expressing E1 or c-Myc-tagged E2 were homogenized in a "vesicular buffer" (containing 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 mg/ml BSA, and protease inhibitor mixture) with a Teflon glass homogenizer.…”

“…has been shown that vesicles containing transmembrane channels or receptors injected into oocytes can fuse with oocyte cell membrane, leading to an incorporation of functional channels or receptors in the oocyte surface membrane (38,39). Injecting vesicles prepared from E1-expressing COS-7 cells (E1 vesicles) into control oocytes (expressing endogenous xQ1 (3)) produced a typical I Ks current within a few hours, and the current was stable for up to 22 h (Fig.…”

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in <em>Saccharomyces cerevisiae</em>

“…We have previously reported a protocol for the large-scale expression of GFP-and His-tagged murine CFTR in S. cerevisiae [15][16][17][18][19][20][21] . The ionic detergent 1-tetradecanoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (LPG-14) is highly efficient in the solubilization of CFTR and has previously been used in the purification of functional membrane proteins 10,22,23 , including purification of CFTR from S. cerevisiae 24 .…”