SummaryIon channels play key roles in cell membranes, and recent advances are yielding an increasing number of structures. However, their functional relevance is often unclear and better tools are required for their functional annotation. In sub-nanometer pores such as ion channels, hydrophobic gating has been shown to promote dewetting to produce a functionally closed (i.e., non-conductive) state. Using the serotonin receptor (5-HT3R) structure as an example, we demonstrate the use of molecular dynamics to aid the functional annotation of channel structures via simulation of the behavior of water within the pore. Three increasingly complex simulation analyses are described: water equilibrium densities; single-ion free-energy profiles; and computational electrophysiology. All three approaches correctly predict the 5-HT3R crystal structure to represent a functionally closed (i.e., non-conductive) state. We also illustrate the application of water equilibrium density simulations to annotate different conformational states of a glycine receptor.

Nanopores in membranes have a range of potential applications. Biomimetic design of nanopores aims to mimic key functions of biological pores within a stable template structure. Molecular dynamics simulations have been used to test whether a simple β-barrel protein nanopore can be modified to incorporate a hydrophobic barrier to permeation. Simulations have been used to evaluate functional properties of such nanopores, using water flux as a proxy for ionic conductance. The behavior of these model pores has been characterized as a function of pore size and of the hydrophobicity of the amino acid side chains lining the narrow central constriction of the pore. Potential of mean force calculations have been used to calculate free energy landscapes for water and for ion permeation in selected models. These studies demonstrate that a hydrophobic barrier can indeed be designed into a β-barrel protein nanopore, and that the height of the barrier can be adjusted by modifying the number of consecutive rings of hydrophobic side chains. A hydrophobic barrier prevents both water and ion permeation even though the pore is sterically unoccluded. These results both provide insights into the nature of hydrophobic gating in biological pores and channels, and furthermore demonstrate that simple design features may be computationally transplanted into β-barrel membrane proteins to generate functionally complex nanopores.

It is desirable that nanopores that are components of biosensors are gated, i.e., capable of controllable switching between closed (impermeable) and open (permeable) states. A central hydrophobic barrier within a nanopore may act as a voltage-dependent gate via electrowetting, i.e., changes in nanopore surface wettability by application of an electric field. We use "computational electrophysiology" simulations to demonstrate and characterize electrowetting of a biomimetic nanopore containing a hydrophobic gate. We show that a hydrophobic gate in a model β-barrel nanopore can be functionally opened by electrowetting at voltages that do not electroporate lipid bilayers. During the process of electrowetting, voltage-induced alignment of water dipoles occurs within the hydrophobic gate region of the nanopore, with water entry preceding permeation of ions through the opened nanopore. When the ionic imbalance that generates a transbilayer potential is dissipated, water is expelled from the hydrophobic gate and the nanopore recloses. The open nanopore formed by electrowetting of a "featureless" β-barrel is anionic selective due to the transmembrane dipole potential resulting from binding of Na ions to the headgroup regions of the surrounding lipid bilayer. Thus, hydrophobic barriers can provide voltage-dependent gates in designed biomimetic nanopores. This extends our understanding of hydrophobic gating in synthetic and biological nanopores, providing a framework for the design of functional nanopores with tailored gating functionality.

A macromolecular nanopore inserted into a membrane may perturb the dynamic organization of the surrounding lipid bilayer. To better understand the nature of such perturbations, we have undertaken a systematic molecular dynamics simulation study of lipid bilayer structure and dynamics around three different classes of nanopore: a carbon nanotube, three related cyclic peptide nanotubes differing in the nature of their external surfaces, and a model of a β-barrel nanopore protein. Periodic spatial distributions of several lipid properties as a function of distance from the nanopore were observed. This was especially clear for the carbon nanotube system, for which the density of lipids, the bilayer thickness, the projection of lipid head-to-tail vectors onto the membrane plane, and lipid lateral diffusion coefficients exhibited undulatory behavior as a function of the distance from the surface of the channel. Overall, the differences in lipid behavior as a function of the nanopore structure reveal local adaptation of the bilayer structure and dynamics to different embedded nanopore structures. Both the local structure and dynamic behavior of lipids around membrane-embedded nanopores are sensitive to the geometry and nature of the outer surface of the macromolecule/molecular assembly forming the pore.

Gating in channels and nanopores plays a key role in regulating flow of ions across membranes. Molecular simulations provide a 'computational microscope' which enables us to examine the physical nature of gating mechanisms at the level of the single channel molecule. Water enclosed within the confines of a nanoscale pore may exhibit unexpected behaviour. In particular, if the molecular surfaces lining the pore are hydrophobic this promotes de-wetting of the pore. De-wetting is observed as stochastic liquid-vapour transitions within a pore, and may lead to functional closure of a pore to the flow of ions and/or water. Such behaviour was first observed in simulations of simple model nanopores and referred to as 'hydrophobic gating'. Simulations of both the nicotinic acetylcholine receptor and of TWIK-1 potassium channels (the latter alongside experimental studies) suggest hydrophobic gating may occur in some biological ion channels. Current studies are focused on designing hydrophobic gates into biomimetic nanopores.