Structural Dynamics of Potassium Channel Monomer in a Membrane Environment and Tetrameric Assembly

Abstract

The folding of a potassium channel monomer and its assembly into a tetramer was investigated using a multi-disciplinary approach combining computations and experiments. Molecular dynamics (MD) simulations of an isolated subunit of the pore domain of the Kv1.2 and KcsA channels in a lipid bilayer were carried out, followed by a Markov State Model (MSM) analysis. The results show that the monomer of these channels can adopt a very wide range of conformations while the two transmembrane (TM) helices and the pore helix retain their secondary structure. This view is supported by NMR spectroscopy showing that the TM helices of single monomers of the KcsA channel embedded in nanodisc or bicelle are partially disordered. A KcsA mutant construct with a disulphide bridge engineered between the two TM helices (A29C-A109C) shows an HSQC spectrum with well-dispersed peaks and limited heterogeneity on NMR time-scale, indicating that the monomer can be locked into a “native-like” conformation by reducing the disorder of the 2 TMs helices. The refolding and assembly of the tetrameric channel in liposomes is examined using a gel-based refolding assay and fluorescent energy transfer (FRET) experiments. The results suggest that the wild-type monomers are partially disordered in the bilayer, with a high proclivity to misfold due to stable non-native helical tetramerization, and that re-folding is limited by a slow concentration-independent unimolecular event. We speculate that the slow step could correspond to the re-orientation of the monomers and the insertion of the pore helix into a disordered tetrameric assembly of 8 TM helices.