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

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.