EPR Studies of Gating Mechanisms in Ion Channels

Abstract

Ion channels open and close in response to diverse stimuli, and the molecular events underlying these processes are extensively modulated by ligands of both endogenous and exogenous origin. In the past decade, high-resolution structures of several channel types have been solved, providing unprecedented details of the molecular architecture of these membrane proteins. Intrinsic conformational flexibility of ion channels critically governs their functions. However, the dynamics underlying gating mechanisms and modulations are obscured in the information from crystal structures. While nuclear magnetic resonance spectroscopic methods allow direct measurements of protein dynamics, they are limited by the large size of these membrane protein assemblies in detergent micelles or lipid membranes. Electron paramagnetic resonance (EPR) spectroscopy has emerged as a key biophysical tool to characterize structural dynamics of ion channels and to determine stimulus-driven conformational transition between functional states in a physiological environment. This review will provide an overview of the recent advances in the field of voltage- and ligand-gated channels and highlight some of the challenges and controversies surrounding the structural information available. It will discuss general methods used in site-directed spin labeling and EPR spectroscopy and illustrate how findings from these studies have narrowed the gap between high-resolution structures and gating mechanisms in membranes, and have thereby helped reconcile seemingly disparate models of ion channel function.

The Role of Proton Transport in Gating Current in a Voltage Gated Ion Channel, as Shown by Quantum Calculations

“…The technique was described by Perozo3 et al in 2002 [ 63 , 64 ]. There have been a number of applications, especially, but not exclusively, to ligand gated channels [ 65 , 66 ]. However, Li et al studied the VSD associated with the Ci-VSP, using both X-ray and EPR [ 67 ]; here the VSD is fairly similar to that of the potassium channels.…”

On the Use of Q-Band Double Electron–Electron Resonance To Resolve the Relative Orientations of Two Double Histidine-Bound Cu2+ Ions in a Protein

“…Distance measurements by pulsed electron paramagnetic resonance (EPR) have become important techniques for the determination of macromolecular structure and dynamics. , These pulsed EPR techniques isolate the weak dipolar coupling between two unpaired electrons in a macromolecule and extract a distance in the range of 2–16 nm. − Such long-range distance constraints are useful to characterize the structure and flexibility of the macromolecule. Specifically, one exciting application of these EPR distance measurements is the elucidation of induced conformational changes in biomolecules. ,,− Such EPR techniques are particularly advantageous for biomolecules that are difficult to crystallize or too large for NMR structural determination.Therefore, pulsed EPR can provide unprecedented insight into the structure and conformations of important biomolecules that would be inaccessible by other means, leading to a greater understanding of the mechanisms of biological processes.…”

A chimeric prokaryotic pentameric ligand–gated channel reveals distinct pathways of activation

“…The prokaryotic homologues, GLIC and ELIC, have served as excellent structural surrogates in describing protein motions underlying channel gating and in identifying conserved drug-binding regions. In GLIC, details of the conformational changes associated with channel activation come from crystal structures of the channel in the putative open and closed conformations ( Bocquet et al, 2009 ; Hilf and Dutzler, 2009 ; Sauguet et al, 2013 , 2014 ) and from electron paramagnetic resonance (EPR) spectroscopy studies of the channel embedded in membranes ( Velisetty et al, 2012 , 2014 ; Dellisanti et al, 2013 ; Chakrapani, 2015 ). Collectively, these studies reveal that during transitions from the closed to the open/desensitized states, there are extensive changes in quaternary structure, domain interfaces, solvent and lipid accessibility, and overall channel dynamics.…”