Investigation of the Binding of the Human KCNE1 Protein with the Voltage Gated Potassium Channel KCNQ1 using DEER EPR Spectroscopy

Abstract

Voltage-gated ion channels are essential for the electrical excitability of neurons, muscles and other excitable cells. KCNE1 is a single transmembrane protein that modulates the activity of voltage gated potassium ion channels (Kv). In the human heart, KCNE1 (E1) interacts with KCNQ1 (Q1) and decreases the rate of channel activation, increases conductance, and generates a slowly activating K+ current critical for cardiac repolarization. Mutations on either KCNE1 or KCNQ1 genes in E1/Q1 complex can lead to cardiac long QT syndrome. Despite the biological significance of the Q1/E1 interaction, its exact nature is not fully understood. In this study, we have used electron paramagnetic resonance (EPR) experiments and double electron electron resonance (DEER) distance measurements to provide the direct evidence of binding activities of KCNQ1 with KCNE1. CW-EPR titration experiment performed on different sites of the C-terminus of KCNQ1 indicated that the EPR lineshape broadening increases with the addition of WT-KCNE1 and saturates at the molar ratio of 1:3 (Q1:E1) suggesting that the spin label motion on Q1 decreases, while interacting with E1. DEER distance measurements on the doubly spin labeled Q1 (374/395 and 524/540) change while the distance measurement on the doubly spin labeled Q1 (445/460) remains the same upon addition of WT-KCNE1 indicating that the regions close to sites 374/395 and 524/540 are interacting strongly with KCNE1. DEER was also used to measure distances between the spin labels attached on E1 and Q1 separately in the Q1/E1 complex to determine the interacting sites in more detail. These results provide direct evidence of binding of Q1 with E1 and will be very useful for determining the structural model of the Q1/E1 complex.