KCNE3 (E3) is a β-subunit of voltage gated potassium ion channels (Kv) that modulates the function of Kv by interacting with its a subunits including KCNQ1 and KCNQ4 (known as Q1 and Q4). Compared to other Kv channels, E3-Q1 interactions have been well studied in intestinal and tracheal epithelial cells where the complex maintains ion homeostasis. E3 interaction with Q1 allows Ky to act as a voltage independent and constitutively active channel that facilitates transepithelial Cl- ion secretion. Mutations in KCNE3 are responsible for diseases related to salt and fluid disorders including cystic fibrosis. Hence, it is important to elucidate KCNE3 structural and dynamic information with functional studies. Previously, the structure of E3 has been studied in a bicelle environment. However, in physiological conditions, lipids are organized into bilayers instead of single sheets of molecules. This research has been focused on protein structure, dynamic and topology of KCNE3 membrane protein in POPC/POPG vesicle usingSite-Directed Spin Labeling (SDSL) and Electron Paramagnetic Resonance Spectroscopy (EPR)techniques. EPR coupled with SDSL provides structural and dynamic information about proteins. In this study we have successfully introduced S-(2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl) methyl methanesulfonothioate spin label (MTSL) on several site-specific position of E3 by site-directed mutagenesis to obtain EPR spectra. CW-EPR (Continuous Wave Electron Paramagnetic Resonance)line shape analysis has been performed to investigate the dynamic of the protein in a lipid bilayer, while EPR power saturation has been used to study the topology of KCNE3 in a vesicle. Our data are consistent with the model of KCNE3 in a bicelle and revealed more promising structure and dynamic information for the protein in a native membrane environment.