The control of ion channel permeation requires the modulation of energetic barriers or “gates” within their pores. However, such barriers are often simply identified from the physical dimensions of the pore. Such approaches have worked well in the past, but there is now evidence that the unusual behavior of water within narrow hydrophobic pores can produce an energetic barrier to permeation without requiring steric occlusion of the pathway. Many different ion channels have now been shown to exploit “hydrophobic gating” to regulate ion flow, and it is clear that new tools are required for more accurate functional annotation of the increasing number of ion channel structures becoming available. We have previously shown how molecular dynamics simulations of water can be used as a proxy to predict hydrophobic gates, and we now present a new and highly versatile computational tool, the Channel Annotation Package (CHAP) that implements this methodology.

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.

Ion channel proteins form water-filled nanoscale pores within lipid bilayers, and their properties are dependent on the complex behavior of water in a nanoconfined environment. Using a simplified model of the pore of the 5-HT 3 receptor (5HT3R) which restrains the backbone structure to that of the parent channel protein from which it is derived, we compare additive with polarizable models in describing the behavior of water in nanopores. Molecular dynamics simulations were performed with four conformations of the channel: two closed state structures, an intermediate state, and an open state, each embedded in a phosphatidylcholine bilayer. Water density profiles revealed that for all water models, the closed and intermediate states exhibited strong dewetting within the central hydrophobic gate region of the pore. However, the open state conformation exhibited varying degrees of hydration, ranging from partial wetting for the TIP4P/2005 water model to complete wetting for the polarizable AMOEBA14 model. Water dipole moments calculated using polarizable force fields also revealed that water molecules remaining within dewetted sections of the pore resemble gas phase water. Free energy profiles for Na + and for Cl − ions within the open state pore revealed more rugged energy landscapes using polarizable force fields, and the hydration number profiles of these ions were also sensitive to induced polarization resulting in a substantive reduction of the number of waters within the first hydration shell of Cl − while it permeates the pore. These results demonstrate that induced polarization can influence the complex behavior of water and ions within nanoscale pores and provides important new insights into their chemical properties.

Ion channel proteins control ionic flux across biological membranes through conformational changes in their transmembrane pores. An exponentially increasing number of channel structures captured in different conformational states are now being determined; however, these newly resolved structures are commonly classified as either open or closed based solely on the physical dimensions of their pore, and it is now known that more accurate annotation of their conductive state requires additional assessment of the effect of pore hydrophobicity. A narrow hydrophobic gate region may disfavor liquid-phase water, leading to local dewetting, which will form an energetic barrier to water and ion permeation without steric occlusion of the pore. Here we quantify the combined influence of radius and hydrophobicity on pore dewetting by applying molecular dynamics simulations and machine learning to nearly 200 ion channel structures. This allows us to propose a simple simulation-free heuristic model that rapidly and accurately predicts the presence of hydrophobic gates. This not only enables the functional annotation of new channel structures as soon as they are determined, but also may facilitate the design of novel nanopores controlled by hydrophobic gates.

Simulations of water behaviour have been used to probe hydrophobic gates in BEST1 and TMEM175, which can reveal important design principles for the engineering of gates in novel biomimetic nanopores.