TREK-2 (KCNK10/K2P10), a two-pore domain potassium (K2P) channel, is gated by multiple stimuli such as stretch, fatty acids, and pH and by several drugs. However, the mechanisms that control channel gating are unclear. Here we present crystal structures of the human TREK-2 channel (up to 3.4 angstrom resolution) in two conformations and in complex with norfluoxetine, the active metabolite of fluoxetine (Prozac) and a state-dependent blocker of TREK channels. Norfluoxetine binds within intramembrane fenestrations found in only one of these two conformations. Channel activation by arachidonic acid and mechanical stretch involves conversion between these states through movement of the pore-lining helices. These results provide an explanation for TREK channel mechanosensitivity, regulation by diverse stimuli, and possible off-target effects of the serotonin reuptake inhibitor Prozac.

Biological ion channels are nanoscale transmembrane pores. When water and ions are enclosed within the narrow confines of a sub-nanometer hydrophobic pore, they exhibit behavior not evident from macroscopic descriptions. At this nanoscopic level, the unfavorable interaction between the lining of a hydrophobic pore and water may lead to stochastic liquid-vapor transitions. These transient vapor states are "dewetted", i.e. effectively devoid of water molecules within all or part of the pore, thus leading to an energetic barrier to ion conduction. This process, termed "hydrophobic gating", was first observed in molecular dynamics simulations of model nanopores, where the principles underlying hydrophobic gating (i.e., changes in diameter, polarity, or transmembrane voltage) have now been extensively validated. Computational, structural, and functional studies now indicate that biological ion channels may also exploit hydrophobic gating to regulate ion flow within their pores. Here we review the evidence for this process and propose that this unusual behavior of water represents an increasingly important element in understanding the relationship between ion channel structure and function.

Synapse formation requires interactions between pre- and postsynaptic cells to establish the connection of a presynaptic nerve terminal with the neurotransmitter receptor-rich postsynaptic apparatus. At developing vertebrate neuromuscular junctions, acetylcholine receptor (AChR) clusters of nascent postsynaptic apparatus are not apposed by presynaptic nerve terminals. Two opposing activities subsequently promote the formation of synapses: positive signals stabilize the innervated AChR clusters, whereas negative signals disperse those that are not innervated. Although the nerve-derived protein agrin has been suggested to be a positive signal, the negative signals remain elusive. Here, we show that cyclin-dependent kinase 5 (Cdk5) is activated by ACh agonists and is required for the ACh agonist-induced dispersion of the AChR clusters that have not been stabilized by agrin. Genetic elimination of Cdk5 or blocking ACh production prevents the dispersion of AChR clusters in agrin mutants. Therefore, we propose that ACh negatively regulates neuromuscular synapse formation through a Cdk5-dependent mechanism.

Ethanol modifies neural activity in the brain by modulating ion channels. Ethanol activates G protein-gated inwardly rectifying K+ channels, but the molecular mechanism is not well understood. Here, we used a crystal structure of a mouse inward rectifier containing a bound alcohol and structure-based mutagenesis to probe a putative alcohol-binding pocket located in the cytoplasmic domains of GIRK channels. Substitutions with bulkier side-chains in the alcohol-binding pocket reduced or eliminated activation by alcohols. By contrast, alcohols inhibited constitutively open channels, such as IRK1 or GIRK2 that binds PIP2 strongly. Mutations in the hydrophobic alcohol-binding pocket of these channels had no effect on alcohol-dependent inhibition, suggesting an alternate site is involved in inhibition. Comparison of high-resolution structures of inwardly rectifying K+ channels suggests a model for activation of GIRK channels utilizing this hydrophobic alcohol-binding pocket. These results provide a tool for developing therapeutic compounds that could mitigate the effects of alcohol.