Probing the Structure and Binding of KCNE1 to the Voltage-Gated Potassium Channel KCNQ1 using Pulsed EPR Spectroscopy

Abstract

Pulsed Electron Paramagnetic Resonance (EPR) spectroscopic techniques coupled with site-directed spin-labeling (SDSL) can provide important structural information on complicated biological systems such as membrane proteins. We are developing EPR techniques such as Double Electron-Electron Resonance (DEER) and Electron Spin Echo Envelope Modulation (ESEEM) for studying membrane proteins. 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 long QT syndrome. Despite the biological significance of the Q1/E1 interaction, its exact nature is not fully understood. CW-EPR, ESEEM and DEER measurements will be used to probe the structure and binding of KCNQ1 with KCNE1. ESEEM spectroscopy was used to probe the secondary structure of different regions of 2H labeled E1. 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 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.