Oliver Beckstein's DPhil Thesis

The directory contains my thesis as a PDF file. In fact, I keep fixing small mistakes that I find over the years. The revision is indicated on the first front matter page. Previous revisions are kept here for archival purposes. The latest version is always OB_thesis_2sided.pdf.

Principles of Gating Mechanisms of Ion Channels

Oliver Beckstein
Laboratory of Molecular Biophysics and Merton College, Oxford
Submitted for the degree of Doctor of Philosophy, Michaelmas 2004
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Ion channels such as the nicotinic acetylcholine receptor (nAChR) fulfil essential roles in fast nerve transmission and cell signalling by converting an external signal into an ionic current, which in turn triggers further down-stream signalling events in the cell. Increasing structural evidence suggests that the actual mechanisms by which channels gate (i.e. switch their ion permeability) are fairly universal: Conduction pathways are either physically occluded by localised sidechains or the pore is narrowed by large-scale protein motions so that a constriction lined by hydrophobic sidechains is formed. In this work the latter mechanism, termed hydrophobic gating, is investigated by atomistic computer simulations.

Simple hydrophobic model pores were constructed with dimensions estimated for the putative gate region of nAChR (length 0.8 nm, radius varied between 0.15 nm and 1.0 nm). In long classical molecular dynamics (MD) simulations, water confined in the pore was found to oscillate between a liquid and a vapour phase on a nano second time scale. Water would rarely permeate a pore less wide than three water molecules. A simple thermodynamic model based on surface energies was developed, which explains the observed liquid-vapour oscillations and their dependence on pore radius and surface hydrophobicity. Similarly, sodium ion flux is only appreciable for pore radii greater than 0.6 nm. Calculation of the free energy profile of translocating ions showed barriers to permeation of greater 10 kT for pore radii less than 0.4 nm. Comparison to continuum-electrostatic Poisson-Boltzmann calculations indicates that the behaviour of the solvent, i.e. water, is crucial for a correct description of ions in apolar pores. Together, these results indicate that a hydrophobic constriction site can act as a hydrophobic gate.

An ongoing debate concerns the nature and position of the gate in nAChR. Based on the recent cryo-electron microscopy structure of the transmembrane domain at 4 Å resolution, and using techniques established for the model pores, equilibrium densities and free energy profiles were calculated for Na+, Cl-, and water. It was found that ions would have to overcome a sizable free energy barrier of about 10 kT at a hydrophobic girdle between residues L9' and V13', previously implicated in gating. This suggests strongly that nAChR contains a hydrophobic gate. Furthermore, charged rings at both ends of the pore act as concentrators of ions up to about six times the bulk concentration; an effect which would increase the ion current in the open state. The robustness of the results is discussed with respect to different parameter sets (force fields) and the applied modelling procedure.


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