Solution-Based Single-Molecule FRET Studies of K+ Channel Gating in a Lipid Bilayer

Emma E Sadler,Achillefs N Kapanidis,Stephen J Tucker

doi:10.1016/j.bpj.2016.05.020

Emma E Sadler, Achillefs N Kapanidis + Show 1 more

Open Access

https://doi.org/10.1016/j.bpj.2016.05.020

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Journal: Biophysical journal	Publication Date: Jun 1, 2016
Citations: 21	License type: cc-by

Affiliation: University of Oxford

Abstract

Ion channels are dynamic multimeric proteins that often undergo multiple unsynchronized structural movements as they switch between their open and closed states. Such structural changes are difficult to measure within the context of a native lipid bilayer and have often been monitored via macroscopic changes in Förster resonance energy transfer (FRET) between probes attached to different parts of the protein. However, the resolution of this approach is limited by ensemble averaging of structurally heterogeneous subpopulations. These problems can be overcome by measurement of FRET in single molecules, but this presents many challenges, in particular the ability to control labeling of subunits within a multimeric protein with acceptor and donor fluorophores, as well as the requirement to image large numbers of individual molecules in a membrane environment. To address these challenges, we randomly labeled tetrameric KirBac1.1 potassium channels, reconstituted them into lipid nanodiscs, and performed single-molecule FRET confocal microscopy with alternating-laser excitation as the channels diffused in solution. These solution-based single-molecule FRET measurements of a multimeric ion channel in a lipid bilayer have allowed us to probe the structural changes that occur upon channel activation and inhibition. Our results provide direct evidence of the twist-to-shrink movement of the helix bundle crossing during channel gating and demonstrate how this method might be applied to real-time structural studies of ion channel gating.

Highlights

Ion channels are complex multimeric structures, and understanding how they open and close represents a major technical challenge [1,2,3]
The approach we describe here exploits the use of nanodiscs to solubilize the channels, thereby allowing alternating-laser excitation (ALEX) confocal-in-solution microscopy to be combined with Single-molecule FRET (smFRET) [8,9]
Since wild-type KirBac1.1 has no endogenous cysteine residues, single-labeling sites were introduced into either the second transmembrane (TM) helix (R151C in TM2) just below the helix bundle crossing (HBC) or at a position (G249C) in the C-terminal domain (CTD) that is well separated from the membrane-embedded region (Fig. 1)

Summary

Introduction

Ion channels are complex multimeric structures, and understanding how they open and close represents a major technical challenge [1,2,3]. The relative stability of these different states can be highly dependent upon the lipid environment, which is often disturbed upon detergent solubilization; for example, once isolated from the membrane, some channels preferentially crystallize in the closed state and have to be forced into the open state by mutagenesis [4,5,6]. These problems are in marked contrast to functional studies of ion channels, which for many decades have employed electrophysiological methods to obtain high-resolution data from single ion channels in a native membrane environment [7]. Better methods for dynamic structural studies of single ion chan-

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