Reconstitution of KCNE1 into Lipid Bilayers: Comparing the Structural, Dynamic, and Activity Differences in Micelle and Vesicle Environments

Abstract

KCNE1 (minK), found in the human heart and, cochlea is a transmembrane protein that modulates the voltage-gated potassium KCNQ1 channel. While KCNE1 has previously been the subject of extensive structural studies in lyso-phospholipid detergent micelles, key observations have yet to be confirmed and refined in lipid bilayers. In this study a reliable method for reconstituting KCNE1 into lipid bilayer vesicles composed of POPC and POPG was developed. Microinjection of the proteoliposomes into Xenopus oocytes expressing the human KCNQ1 (KV7.1) voltage-gated potassium channel led to native-like modulation of the channel. CD spectroscopy demonstrated that the %-helicity of KCNE1 is significantly higher for the protein reconstituted in lipid vesicles relative to the previously described structure in 1.0% LMPG micelles. SDSL EPR spectroscopic techniques were used to probe the local structure and environment of Ser28, Phe54, Phe57, Leu 59, and Ser64 KCNE1 in both POPC/POPG vesicles and in LMPG micelles. Spin-labeled KCNE1 cysteine mutants at Phe54, Phe57, Leu 59, and Ser64 were found to be located inside POPC/POPG vesicles, whereas Ser28 was found to be located outside the membrane. Ser64 was shown to be water-inaccessible in vesicles, but found to be water-accessible in LMPG micelle solutions. These results suggest that key components of the micelle-derived structure of KCNE1 extend to the structure of this protein in lipid bilayers, but also demonstrates the need to refine this structure using data derived from bilayer-reconstituted protein in order to more accurately define its native structure. This work establishes the basis for such future studies.

Probing Structural Dynamics and Topology of the KCNE1 Membrane Protein in Lipid Bilayers via Site-Directed Spin Labeling and Electron Paramagnetic Resonance Spectroscopy

“…However, the second choice is less likely because most native membrane systems contain a mixture of PC and PG lipids. 12,15,16,29 …”

“…2 POPC/POPG lipids were used for this study since they are typically found in mammalian membranes. 12,15,16,29 …”

“…2,7,52 The structural topology and dynamics model reported in this study are consistent with the POPC/POPG lipid bilayer width and their membrane surface properties. 12,13 However, the depth parameter values and dynamic flexibility may vary depending upon the length and choice of phospholipids used for the measurements. 53 Since the extracellular domain of KCNE1 undergoes two major conformations (as evidenced by two major CW-EPR spectral components, Figure 2), the EPR power saturation depth parameters corresponding to the sites in this domain might reflect the average saturation behavior of the multiple components.…”

“…12 Samples were loaded into gas permeable TPX capillary tubes with a total volume of 3–4 μ L at a concentration of 40–60 μ M. EPR data collection was carried out using a modulation amplitude of 1 G and a varying microwave power of 0.4–100 mW. The scan range of all spectra was 100 G, and the final spectra were obtained by signal averaging 10 scans.…”

Characterizing the structure of lipodisq nanoparticles for membrane protein spectroscopic studies

“…Here, we use POPC/POPG lipids at a molar ratio of 9/1 and SMA polymers to form lipodisq nanoparticles. POPC is the most common phospholipid found in mammalian cell membranes, and POPG was chosen to mimic the 10 – 20 molar percent anionic phospholipids typically found in mammalian membranes 2829 . The weight ratio of lipid to polymer was varied and different lipodisq nanoparticle samples were characterized by solid-state NMR (SSNMR) and dynamic light scattering (DLS).…”