General anaesthetics have enjoyed long and widespread use but their molecular mechanism of action remains poorly understood. There is good evidence that their principal targets are pentameric ligand-gated ion channels (pLGICs) such as inhibitory GABA(A) (γ-aminobutyric acid) receptors and excitatory nicotinic acetylcholine receptors, which are respectively potentiated and inhibited by general anaesthetics. The bacterial homologue from Gloeobacter violaceus (GLIC), whose X-ray structure was recently solved, is also sensitive to clinical concentrations of general anaesthetics. Here we describe the crystal structures of the complexes propofol/GLIC and desflurane/GLIC. These reveal a common general-anaesthetic binding site, which pre-exists in the apo-structure in the upper part of the transmembrane domain of each protomer. Both molecules establish van der Waals interactions with the protein; propofol binds at the entrance of the cavity whereas the smaller, more flexible, desflurane binds deeper inside. Mutations of some amino acids lining the binding site profoundly alter the ionic response of GLIC to protons, and affect its general-anaesthetic pharmacology. Molecular dynamics simulations, performed on the wild type (WT) and two GLIC mutants, highlight differences in mobility of propofol in its binding site and help to explain these effects. These data provide a novel structural framework for the design of general anaesthetics and of allosteric modulators of brain pLGICs.

Neurotransmitters such as acetylcholine (ACh) and glycine mediate fast synaptic neurotransmission by activating pentameric ligandgated ion channels (LGICs). These receptors are allosteric transmembrane proteins that rapidly convert chemical messages into electrical signals. Neurotransmitters activate LGICs by interacting with an extracellular agonist-binding domain (ECD), triggering a tertiary͞quaternary conformational change in the protein that results in the fast opening of an ion pore domain (IPD). However, the molecular mechanism that determines the fast opening of LGICs remains elusive. Here, we show by combining whole-cell and single-channel recordings of recombinant chimeras between the ECD of ␣7 nicotinic receptor (nAChR) and the IPD of the glycine receptor (GlyR) that only two GlyR amino acid residues of loop 7 (Cys-loop) from the ECD and at most five ␣7 nAChR amino acid residues of the M2-M3 loop (2-3L) from the IPD control the fast activation rates of the ␣7͞Gly chimera and WT GlyR. Mutual interactions of these residues at a critical pivot point between the agonist-binding site and the ion channel fine-tune the intrinsic opening and closing rates of the receptor through stabilization of the transition state of activation. These data provide a structural basis for the fast opening of pentameric LGICs.allosteric proteins ͉ chimeric receptor ͉ Cys-loop receptor ͉ transition state P entameric ligand-gated ion channels (LGICs), such as the cationic nicotinic acetylcholine receptor (nAChR) and the anionic glycine receptor (GlyR), mediate fast excitatory or inhibitory chemical neurotransmission between neurons (1-6). A unique feature of these receptors is that they activate the ion channel, a process known as gating, in less than a ms. For nicotinic receptors, a detailed single-channel analysis has recently established a speed limit for the opening of the ion channel in the s time range (7). Perturbations of this rapid transmission pathway by natural mutants lead to severe diseases such as congenital myasthenic syndromes (8), hereditary hyperekplexia (9), or epileptic disorders (10).Pentameric LGICs, or Cys-loop receptors, are composed of five homologous subunits, sharing a common structural organization, arranged (pseudo)symmetrically around the central ion pore (1, 2). All subunits are made of two distinct topological domains: the extracellular (ECD) and the ion pore domains (IPD). First, the ECD is folded into a twisted ␤-sandwich core, as revealed by x-ray crystallographic studies of the mollusk acetylcholine-binding protein (AChBP), a soluble pentameric protein homologous to the extracellular domain of LGICs (11-14). Second, electron microscopy images of Torpedo nAChR at 4-Å resolution revealed that the four transmembrane segments (M1 to M4) of the IPD are folded into ␣-helices joined by linking loops of variable lengths (15). By combining these structural data, we built a 3D model of the full ␣7 nAChR (16). In this model, the coupling zone located at the interface between the two domains is framed by f...

Pentameric ligand-gated ion channels (pLGICs) are widely expressed in the animal kingdom and are key players of neurotransmission by acetylcholine (ACh), γ-amminobutyric acid (GABA), glycine and serotonin. It is now established that this family has a prokaryotic origin, since more than 20 homologues have been discovered in bacteria. In particular, the GLIC homologue displays a ligand-gated ion channel function and is activated by protons. The prokaryotic origin of these membrane proteins facilitated the X-ray structural resolution of the first members of this family. ELIC was solved at 3.3Å in a closed-pore conformation, and GLIC at up to 2.9Å in an apparently open-pore conformation. These data reveal many structural features, notably the architecture of the pore, including its gate and its selectivity filter, and the interactions between the protein and lipids. In addition, comparison of the structures of GLIC and ELIC hints at a mechanism of channel opening, which consists of both a quaternary twist and a tertiary deformation. This mechanism couples opening-closing motions of the channel with a global reorganization of the protein, including the subunit interface that holds the neurotransmitter binding sites in eukaryotic pLGICs.

ion channel ͉ membrane protein ͉ structure ͉ acetylcholine

No abstract

To learn about the mechanism of ion charge selectivity by invertebrate glutamate-gated chloride (GluCl) channels, we swapped segments between the GluCl␤ receptor of Caenorhabditis elegans and the vertebrate cationic ␣7-acetylcholine receptor and monitored anionic/cationic permeability ratios. Complete conversion of the ion charge selectivity in a set of receptor microchimeras indicates that the selectivity filter of the GluCl␤ receptor is created by a sequence connecting the first with the second transmembrane segments. A single substitution of a negatively charged residue within this sequence converted the selectivity of the GluCl␤ receptor's pore from anionic to cationic. Unexpectedly, elimination of the charge of each basic residue of the selectivity filter, one at a time or concomitantly, moderately reduced the P Cl /P Na ratios, but the GluCl␤ receptor's mutants retained high capacity to select Cl The invertebrate GluCl 2 receptor channels are pentameric transmembrane receptors belonging to a wide superfamily of Cys-loop receptors activated by various neurotransmitters such as acetylcholine (ACh), serotonin (5-hydroxtryptamine, 5HT), ␥-aminobutyric acid (GABA), Gly, Glu, or histamine (Fig. 1A) (1-8). This superfamily consists of cationic channels permeable to Na ϩ , K ϩ , and, in many subunit combinations, to Ca 2ϩ ions, as well as of anionic channels selective to Cl Ϫ ions (reviewed by Keramidas et al. (9)). Structural similarities shared by Cys-loop receptors enabled swapping of pore sequences between cationic and anionic channels so as to assess the involvement of specific amino acids in ion charge selectivity. It was previously shown that concomitant replacement of the residues at positions Ϫ2Ј, Ϫ1Ј, and 13Ј ( Fig. 1, B and C) of cationic receptors by the residues found at the homologous positions of anionic receptors, and vice versa, lead to conversion of ion charge selectivity (10 -13).Further mutagenesis studies led to the recognition that the different capacities of cationic versus anionic Cys-loop receptors to distinguish between the charge of ions rely on the differences in the amino acid composition at positions Ϫ1Ј and Ϫ2Ј (Fig. 1C) (13-16). The conserved pore-facing Glu residue at position Ϫ1Ј of cationic Cys-loop receptors was further inferred to form, around the axis of ion conduction, a negatively charged ring that plays the key role in cationic selectivity by interacting with cations and repulsing anions (12,13,15,16). Conversely, a conserved arginine at position 0Ј of anionic Cys-loop receptors was inferred to interact with anions and repulse cations. A basic residue at position 0Ј is also typical of all cationic Cys-loop receptors (Fig. 1C and the ligand-gated ion channels data base), but it was suggested that local conformational differences in the M1-M2 connecting segment (M1-M2 loop) orient this basic residue to the pore lumen only in anionic Cys-loop receptors (9, 15). These local conformational differences have been attributed to a proline residue, which is present exclusively at pos...

No abstract

Nicotinic acetylcholine receptors (AChRs) belong to a superfamily of oligomeric proteins that transduce electric signals across the cell membrane on binding of neurotransmitters. These receptors harbor a large extracellular ligand-binding domain directly linked to an ion-conducting channel-forming domain that spans the cell membrane 20 times and considerably extends into the cytoplasm. Thus far, none of these receptor channels has been crystallized in three dimensions. The crystallization of the AChR from Torpedo marmorata electric organs is challenged here in lipidic؊detergent matrices. Detergent-soluble AChR complexed with ␣-bungarotoxin (␣BTx), a polypeptidic competitive antagonist, was purified. The AChR-␣BTx complex was reconstituted in a lipidic matrix composed of monoolein bilayers that are structured in three dimensions. The ␣BTx was conjugated to a photo-stable fluorophore, enabling us to monitor the physical behavior of the receptor-toxin complex in the lipidic matrix under light stereomicroscope, and to freeze fracture regions containing the receptor-toxin complex for visualization under a transmission electron microscope. Conditions were established for forming 2D receptor-toxin lattices that are stacked in the third dimension. 3D AChR nanocrystals were thereby grown inside the highly viscous lipidic 3D matrix. Slow emulsification of the lipidic matrix converted these nanocrystals into 3D elongated thin crystal plates of micrometer size. The latter are stable in detergent-containing aqueous solutions and can currently be used for seeding and epitaxial growth, en route to crystals of appropriate dimensions for x-ray diffraction studies.nicotinic acetylcholine receptor ͉ crystallization ͉ structure T o date, the crystal structures of Ϸ50 integral membrane proteins have been determined at atomic resolution. This result is in stark contrast to their abundance, as they comprise one-third of all gene products. Inherent difficulties with highlevel expression, purification, and, particularly, crystallization of integral membrane proteins account for this situation (1). Receptor channels that selectively regulate transport of ions across the cell membrane are large membrane-spanning oligomers. Among them are the pentameric ligand-gated ion channels (also termed the Cys-loop receptors), which mediate and modulate fast cell-cell communication throughout the nervous system.The first and most extensively studied pentameric ligand-gated ion channel is the acetylcholine receptor (AChR; reviewed in refs. 2-4). The AChR isolated from the electric organs of Torpedo ray is available in milligram amounts suitable for crystallization experiments. It is a pseudosymmetrical glycoprotein of Ϸ290 kDa comprised of five subunits (2␣␤␥␦) and carries two ACh-binding sites at the ␣Ϫ␥ and ␣Ϫ␦ boundaries (2, 3). So far, it was possible to organize this oligomeric protein only in tubular 2D crystals within receptor-rich membrane fragments (5, 6). These crystals enabled the collection of electron diffraction data at 9-Å down to 4-Å r...