A pentameric,ligand-gated ion channel from a bacterium

Commentary to:

Hilf RJ, Dutzler R. X-ray structure of a prokaryotic pentameric ligand-gated ion channel. Nature 2008; 452:375-80.     

Desensitization mechanism in prokaryotic ligand-gated ion channel

Crystal structures of Gloeobacter violaceus ligand-gated ion channel (GLIC), a proton-gated prokaryotic homologue of pentameric ligand-gated ion channel (LGIC) from G. violaceus, have provided high-resolution models of the channel architecture and its role in selective ion conduction and drug binding. However, it is still unclear which functional states of the LGIC gating scheme these crystal structures represent. Much of this uncertainty arises from a lack of thorough understanding of the functional properties of these prokaryotic channels. To elucidate the molecular events that constitute gating, we have carried out an extensive characterization of GLIC function and dynamics in reconstituted proteoliposomes by patch clamp measurements and EPR spectroscopy. We find that GLIC channels show rapid activation upon jumps to acidic pH followed by a time-dependent loss of conductance because of desensitization. GLIC desensitization is strongly coupled to activation and is modulated by voltage, permeant ions, pore-blocking drugs, and membrane cholesterol. Many of these properties are parallel to functions observed in members of eukaryotic LGIC. Conformational changes in loop C, measured by site-directed spin labeling and EPR spectroscopy, reveal immobilization during desensitization analogous to changes in LGIC and acetylcholine binding protein. Together, our studies suggest conservation of mechanistic aspects of desensitization among LGICs of prokaryotic and eukaryotic origin.     

LGICdb: the ligand-gated ion channel database

Ligand-Gated Ion Channels (LGIC) are polymeric transmembrane proteins involved in the fast response to numerous neurotransmitters. All these receptors are formed by homologous subunits and the last two decades revealed an unexpected wealth of genes coding for these subunits. The Ligand-Gated Ion Channel database (LGICdb) has been developed to handle this increasing amount of data. The database aims to provide only one entry for each gene, containing annotated nucleic acid and protein sequences. The repository is carefully structured and the entries can be retrieved by various criteria. In addition to the sequences, the LGICdb provides multiple sequence alignments, phylogenetic analyses and atomic coordinates when available. The database is accessible via the World Wide Web (http://www.pasteur.fr/recherche/banques/LGIC /LGIC.html), where it is continuously updated. The version 16 (September 2000) available for download contained 333 entries covering 34 species.     

Stoichiometry of a ligand-gated ion channel determined by fluorescence energy transfer

We have developed a method to determine the stoichiometry of subunits within an oligomeric cell surface receptor using fluorescently tagged antibodies to the individual subunits and measuring energy transfer between them. Anti-c-Myc monoclonal antibody (mAb 9-E10) derivatized with a fluorophore (europium cryptate, EuK) was used to individually label c-Myc-tagged alpha1-, beta2-, or gamma2-subunits of the hetero-oligomeric gamma-aminobutyric acid (GABAA) receptor in intact cells. The maximal fluorescent signal derived from the alpha1(c-Myc)beta2gamma2 and the alpha1beta2(c-Myc)gamma2 receptors was twice that obtained with alpha1beta2gamma2(c-Myc), suggesting that there are 2x alpha-, 2x beta-, and 1x gamma-subunits in a receptor monomer. This observation was extended using fluorescence energy transfer. Receptors were half-maximally saturated with EuK-anti-c-Myc mAb, and the remaining alpha1(c-Myc) subunits were labeled with excess anti-c-Myc mAb derivatized with the fluorescence energy acceptor, XL665. On exposure to laser light, energy transfer from EuK to XL665 occurred with alpha1(c-Myc)beta2gamma2 and alpha1beta2(c-Myc)gamma2, but no significant energy transfer was observed with alpha1beta2gamma2(c-Myc) receptors, indicating the absence of a second gamma-subunit in a receptor monomer. We confirm that the GABAA receptor subtype, alpha1beta2gamma2, is composed of two copies each of the alpha- and beta-subunits and one copy of the gamma-subunit (i.e. (alpha1)2(beta2)2(gamma2)1) and conclude that this method would have general applicability to other multisubunit cell surface proteins.     

Characterizing ligand-gated ion channel receptors with genetically encoded Ca2++ sensors

We present a cell based system and experimental approach to characterize agonist and antagonist selectivity for ligand-gated ion channels (LGIC) by developing sensor cells stably expressing a Ca(2+) permeable LGIC and a genetically encoded F?rster (or fluorescence) resonance energy transfer (FRET)-based calcium sensor. In particular, we describe separate lines with human α7 and human α4β2 nicotinic acetylcholine receptors, mouse 5-HT(3A) serotonin receptors and a chimera of human α7/mouse 5-HT(3A) receptors. Complete concentration-response curves for agonists and Schild plots of antagonists were generated from these sensors and the results validate known pharmacology of the receptors tested. Concentration-response relations can be generated from either the initial rate or maximal amplitudes of FRET-signal. Although assaying at a medium throughput level, this pharmacological fluorescence detection technique employs a clonal line for stability and has versatility for screening laboratory generated congeners as agonists or antagonists on multiple subtypes of ligand-gated ion channels. The clonal sensor lines are also compatible with in vivo usage to measure indirectly receptor activation by endogenous neurotransmitters.     

Regulation of a pentameric ligand-gated ion channel by a semiconserved cationic lipid-binding site

Pentameric ligand-gated ion channels (pLGICs) are crucial mediators of electrochemical signal transduction in various organisms from bacteria to humans. Lipids play an important role in regulating pLGIC function, yet the structural bases for specific pLGIC-lipid interactions remain poorly understood. The bacterial channel ELIC recapitulates several properties of eukaryotic pLGICs, including activation by the neurotransmitter GABA and binding and modulation by lipids, offering a simplified model system for structure–function relationship studies. In this study, functional effects of noncanonical amino acid substitution of a potential lipid-interacting residue (W206) at the top of the M1-helix, combined with detergent interactions observed in recent X-ray structures, are consistent with this region being the location of a lipid-binding site on the outward face of the ELIC transmembrane domain. Coarse-grained and atomistic molecular dynamics simulations revealed preferential binding of lipids containing a positive charge, particularly involving interactions with residue W206, consistent with cation-π binding. Polar contacts from other regions of the protein, particularly M3 residue Q264, further support lipid binding via headgroup ester linkages. Aromatic residues were identified at analogous sites in a handful of eukaryotic family members, including the human GABA_A receptor ε subunit, suggesting conservation of relevant interactions in other evolutionary branches. Further mutagenesis experiments indicated that mutations at this site in ε-containing GABA_A receptors can change the apparent affinity of the agonist response to GABA, suggesting a potential role of this site in channel gating. In conclusion, this work details type-specific lipid interactions, which adds to our growing understanding of how lipids modulate pLGICs.     

Extracellular protons titrate voltage gating of a ligand-gated ion channel

Cyclic nucleotide–gated channels mediate transduction of light into electric signals in vertebrate photoreceptors. These channels are primarily controlled by the binding of intracellular cyclic GMP (cGMP). Glutamate residue 363 near the extracellular end of the ion selectivity filter interacts with the pore helix and helps anchor the filter to the helix. Disruption of this interaction by mutations renders the channels essentially fully voltage gated in the presence of saturating concentrations of cGMP. Here, we find that lowering extracellular pH makes the channels conduct in an extremely outwardly rectifying manner, as does a neutral glutamine substitution at E363. A pair of cysteine mutations, E363C and L356C (the latter located midway the pore helix), largely eliminates current rectification at low pH. Therefore, this low pH-induced rectification primarily reflects voltage-dependent gating involving the ion selectivity filter rather than altered electrostatics around the external opening of the ion pore and thus ion conduction. It then follows that protonation of E363, like the E363Q mutation, disrupts the attachment of the selectivity filter to the pore helix. Loosening the selectivity filter from its surrounding structure shifts the gating equilibrium toward closed states. At low extracellular pH, significant channel opening occurs only when positive voltages drive the pore from a low probability open conformation to a second open conformation. Consequently, at low extracellular pH the channels become practically fully voltage gated, even in the presence of a saturating concentration of cGMP.     

Ligand activation of the prokaryotic pentameric ligand-gated ion channel ELIC

While the pentameric ligand-gated ion channel ELIC has recently provided first insight into the architecture of the family at high resolution, its detailed investigation was so far prevented by the fact that activating ligands were unknown. Here we describe a study on the functional characterization of ELIC by electrophysiology and X-ray crystallography. ELIC is activated by a class of primary amines that include the neurotransmitter GABA at high micro- to millimolar concentrations. The ligands bind to a conserved site and evoke currents that slowly desensitize over time. The protein forms cation selective channels with properties that resemble the nicotinic acetylcholine receptor. The high single channel conductance and the comparably simple functional behavior make ELIC an attractive model system to study general mechanisms of ion conduction and gating in this important family of neurotransmitter receptors.     

Ion selectivity mechanism in a bacterial pentameric ligand-gated ion channel

The proton-gated ion channel from Gloeobacter violaceus (GLIC) is a prokaryotic homolog of the eukaryotic nicotinic acetylcholine receptor that responds to the binding of neurotransmitter acetylcholine and mediates fast signal transmission. Recent emergence of a high-resolution crystal structure of GLIC captured in a potentially open state allowed detailed, atomic-level insight into ion conduction and selectivity mechanisms in these channels. Herein, we have examined the barriers to ion conduction and origins of ion selectivity in the GLIC channel by the construction of potential-of-mean-force profiles for sodium and chloride ions inside the transmembrane region. Our calculations reveal that the GLIC channel is open for a sodium ion to transport, but presents a ∼11 kcal/mol free energy barrier for a chloride ion. Our collective findings identify three distinct contributions to the observed preference for the permeant ions. First, there is a substantial contribution due to a ring of negatively charged glutamate residues (E-2′) at the narrow intracellular end of the channel. The negative electrostatics of this region and the ability of the glutamate side chains to directly bind cations would strongly favor the passage of sodium ions while hindering translocation of chloride ions. Second, our results imply a significant hydrophobic contribution to selectivity linked to differences in the desolvation penalty for the sodium versus chloride ions in the central hydrophobic region of the pore. This hydrophobic contribution is evidenced by the large free energy barriers experienced by Cl⁻ in the middle of the pore for both GLIC and the E-2′A mutant. Finally, there is a distinct contribution arising from the overall negative electrostatics of the channel.     

The selectivity filter of a ligand-gated ion channel. The helix-M2 model of the ion channel of the nicotinic acetylcholine receptor

Evidence from electrophysiology and biochemistry supports the hypothesis that the ion channel of the nicotinic acetylcholine receptor is formed by homologous amino acid sequences of all receptor subunits, called helices M2. A model of the ion channel is proposed and the selectivity filter is described as a ring of negatively-charged amino acid side chains [(1988) Nature 335, 645-648] which may undergo conformational changes upon permeation of the cation.     

A novel class of Na+ and Ca2+ channel dual blockers with highly potent anti-ischemic effects

A series of novel arylpiperidines (4a-d) which have highly potent blocking effects for both neuronal Na+ and T-type Ca2+ channels with extremely low affinity for dopamine D2 receptors were synthesized. Among these compounds, 1-(2-hydroxy-3-phenoxy)propyl-4-(4-phenoxyphenyl)-piperidine hydrochloride (4c; SUN N5030) exhibited remarkable neuroprotective activity in a transient middle cerebral artery occlusion (MCAO) model.     

Structure of the pore-forming transmembrane domain of a ligand-gated ion channel

The structure of the pore-forming transmembrane domain of the nicotinic acetylcholine receptor from Torpedo has been investigated by infrared spectroscopy. Treatment of affinity-purified receptor with either Pronase or proteinase K digests the extramembranous domains (roughly 75% of the protein mass), leaving hydrophobic membrane-imbedded peptides 3-6 kDa in size that are resistant to peptide (1)H/(2)H exchange. Infrared spectra of the transmembrane domain preparations exhibit relatively sharp and symmetric amide I and amide II band contours centered near 1655 and 1545 cm(-)1, respectively, in both (1)H(2)O and (2)H(2)O. The amide I band is very similar to the amide I bands observed in the spectra of alpha-helical proteins, such as myoglobin and bacteriorhodopsin, that lack beta structure and exhibit much less beta-sheet character than is observed in proteins with as little as 20% beta sheet. Curve-fitting estimates 75-80% alpha-helical character, with the remaining peptides likely adopting extended and/or turn structures at the bilayer surface. Infrared dichroism spectra are consistent with transmembrane alpha-helices oriented perpendicular to the bilayer surface. The evidence strongly suggests that the transmembrane domain of the nicotinic receptor, the most intensively studied ligand-gated ion channel, is composed of five bundles of four transmembrane alpha-helices.     

Noxious cold ion channel TRPA1 is activated by pungent compounds and bradykinin

Six members of the mammalian transient receptor potential (TRP) ion channels respond to varied temperature thresholds. The natural compounds capsaicin and menthol activate noxious heat-sensitive TRPV1 and cold-sensitive TRPM8, respectively. The burning and cooling perception of capsaicin and menthol demonstrate that these ion channels mediate thermosensation. We show that, in addition to noxious cold, pungent natural compounds present in cinnamon oil, wintergreen oil, clove oil, mustard oil, and ginger all activate TRPA1 (ANKTM1). Bradykinin, an inflammatory peptide acting through its G protein-coupled receptor, also activates TRPA1. We further show that phospholipase C is an important signaling component for TRPA1 activation. Cinnamaldehyde, the most specific TRPA1 activator, excites a subset of sensory neurons highly enriched in cold-sensitive neurons and elicits nociceptive behavior in mice. Collectively, these data demonstrate that TRPA1 activation elicits a painful sensation and provide a potential molecular model for why noxious cold can paradoxically be perceived as burning pain.     

Endocytosis function of a ligand-gated ion channel homolog in Caenorhabditis elegans

Ligand-gated ion channels are transmembrane proteins that respond to a variety of transmitters, including acetylcholine, gamma-aminobutyric acid (GABA), glycine, and glutamate [1 and 2]. These proteins play key roles in neurotransmission and are typically found in the nervous system and at neuromuscular junctions [3]. Recently, acetylcholine receptor family members also have been found in nonneuronal cells, including macrophages [4], keratinocytes [5], bronchial epithelial cells [5], and endothelial cells of arteries [6]. The function of these channels in nonneuronal cells in mammals remains to be elucidated, though it has been shown that the acetylcholine receptor alpha7 subunit is required for acetylcholine-mediated inhibition of tumor necrosis factor release by activated macrophages [4]. We show that cup-4, a gene required for efficient endocytosis of fluids by C. elegans coelomocytes, encodes a protein that is homologous to ligand-gated ion channels, with the highest degree of similarity to nicotinic acetylcholine receptors. Worms lacking CUP-4 have reduced phosphatidylinositol 4,5-bisphosphate levels at the plasma membrane, suggesting that CUP-4 regulates endocytosis through modulation of phospholipase C activity.     

An HSV vector system for selection of ligand-gated ion channel modulators

Pathological alterations of ion channel activity result from changes in modulatory mechanisms governing receptor biology. Here we describe a conditional herpes simplex virus (HSV) replication-based strategy to discover channel modulators whereby inhibition of agonist-induced channel activation by a vector-expressed modulatory gene product prevents ion flux, osmotic shock and cell death. Inhibition of channel activity, in this case, the rat vanilloid (Trpv1 or the glycine receptor (GlyRalpha1), can allow selection of escape vector plaques containing the 'captured' modulatory gene for subsequent identification and functional analysis. We validated this prediction using mixed infections of a wild-type Trpv1 expression vector vTTHR and a nonfunctional 'poreless' Trpv1 subunit-expressing vector, vHP, wherein vHP was highly selected from a large background of vTTHR viruses in the presence of the Trpv1 agonist, capsaicin. The approach should be useful for probing large libraries of vector-expressed cDNAs for the presence of ion channel modulators.