The expression, purification, and site-directed spin-labeling of KCNE4

Accessory subunits belonging to the KCNE family modulate voltage-gated potassium (Kv) channels such as Kv1.1, Kv1.3, Kv2.1, and Kv7.1. Incorrect modulation of potassium current through these channels have been linked to several diseases for example, long QT syndrome, allergic rhinitis, and acute lymphoblastic leukemia. Increased understanding of the molecular underpinnings of KCNE modulation of potassium channels would help expand our understanding of the etiology of these diseases. KCNE4 is unique in the KCNE family as it has been shown to inhibit current in Kv channels. Previously, the role of KCNE4's tetraleucine motif has been tied to its inhibitory effects. In this study, we describe a new expression and purification protocol for KCNE4 to enhance our ability to obtain structural and dynamic information using electron paramagnetic resonance (EPR) spectroscopy. Site-directed mutagenesis was used to attach a nitroxide spin-label at residues of interest. This technique in combination with line-shape analysis of continuous-wave EPR spectroscopy (CW-EPR) was used to observe changes in spin-label side-chain mobility as KCNE4 interacts with different membrane mimetic systems. As the spin label on the protein encounters the membrane, the line-shape of CW-EPR spectra broaden indicating a decrease in mobility of the spin label. The expression, purification, and site-directed spin-labeling of KCNE4 developed in this study will be used in the future to determine the role of the tetraleucine motif in protein structure and function to elucidate modulation of Kv channels in native and disease states.