Retrochalcone derivatives are positive allosteric modulators at synaptic and extrasynaptic GABA(A) receptors in vitro.

BACKGROUND AND PURPOSE: Flavonoids, important plant pigments, have been shown to allosterically modulate brain GABA(A) receptors (GABA(A)Rs). We previously reported that trans-6,4'-dimethoxyretrochalcone (Rc-OMe), a hydrolytic derivative of the corresponding flavylium salt, displayed nanomolar affinity for the benzodiazepine binding site of GABA(A)Rs. Here, we evaluate the functional modulations of Rc-OMe, along with two other synthetic derivatives trans-6-bromo-4'-methoxyretrochalcone (Rc-Br) and 4,3'-dimethoxychalcone (Ch-OMe) on GABA(A)Rs. EXPERIMENTAL APPROACH: Whole-cell patch-clamp recordings were made to determine the effects of these derivatives on GABA(A)Rs expressed in HEK-293 cells and in hippocampal CA1 pyramidal and thalamic neurones from rat brain. KEY RESULTS: Rc-OMe strongly potentiated GABA-evoked currents at recombinant α(1-4)ß(2)γ(2s) and α(4)ß(3)δ receptors but much less at α(1)ß(2) and α(4)ß(3). Rc-Br and Ch-OMe potentiated GABA-evoked currents at α(1)ß(2)γ(2s). The potentiation by Rc-OMe was only reduced at α(1)H101Rß(2)γ(2s) and α(1)ß(2)N265Sγ(2s), mutations known to abolish the potentiation by diazepam and loreclezole respectively. The modulation of Rc-OMe and pentobarbital as well as by Rc-OMe and the neurosteroid 3α,21-dihydroxy-5α-pregnan-20-one was supra-additive. Rc-OMe modulation exhibited no apparent voltage-dependence, but was markedly dependent on GABA concentration. In neurones, Rc-Br slowed the decay of spontaneous inhibitory postsynaptic currents and both Rc-OMe and Rc-Br positively modulated synaptic and extrasynaptic diazepam-insensitive GABA(A)Rs. CONCLUSIONS AND IMPLICATIONS: The trans-retrochalcones are powerful positive allosteric modulators of synaptic and extrasynaptic GABA(A)Rs. These novel modulators act through an original mode, thus making them putative drug candidates in the treatment of GABA(A)-related disorders in vivo.

A putative extracellular salt bridge at the subunit interface contributes to the ion channel function of the ATP-gated P2X2 receptor.

The recent crystal structure of the ATP-gated P2X4 receptor revealed a static view of its architecture, but the molecular mechanisms underlying the P2X channels activation are still unknown. By using a P2X2 model based on the x-ray structure, we sought salt bridges formed between charged residues located in a region that directly connects putative ATP-binding sites to the ion channel. To reveal their significance for ion channel activation, we made systematic charge exchanges and measured the effects on ATP sensitivity. We found that charge reversals at the interfacial residues Glu(63) and Arg(274) produced gain-of-function phenotypes that were cancelled upon paired charge swapping. These results suggest that a putative intersubunit salt bridge formed between Glu(63) and Arg(274) contributes to the ion channel function. Engineered cysteines E63C and R274C formed redox-dependent cross-links in the absence of ATP. By contrast, the presence of ATP reduced the rate of disulfide bond formation, indicating that ATP binding might trigger relative movement of adjacent subunits at the level of Glu(63) and Arg(274), allowing the transmembrane helices to open the channel.

Identification of two critical residues within the Cys-loop sequence that determine fast-gating kinetics in a pentameric ligand-gated ion channel.

Pentameric ligand-gated ion channels (LGICs) are fast-gating receptors, represented by cationic nicotinic acetylcholine (nAChR) and serotonin (5HT3R) receptors, and by anionic GABA and glycine (GlyR) receptors. Because of a highly conserved sequence of 13 amino acids flanked by two canonical cysteine residues shared by all members of the family, these receptors are also known as the Cys-loop family. These receptors are allosteric transmembrane proteins made of five identical (or not) subunits arranged (pseudo) symmetrically around a central ion pore in the membrane. In nAChR, upon ACh binding, the receptor interconverts into discrete allosteric states, with each state corresponding to a different physiological state: resting (closed), active (open), and desensitized (closed).

Mechanism of Cl- selection by a glutamate-gated chloride (GluCl) receptor revealed through mutations in the selectivity filter.

To learn about the mechanism of ion charge selectivity by invertebrate glutamate-gated chloride (GluCl) channels, we swapped segments between the GluClbeta receptor of Caenorhabditis elegans and the vertebrate cationic alpha7-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 GluClbeta 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 GluClbeta 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 GluClbeta receptor's mutants retained high capacity to select Cl(-) over Na(+). These results indicate that, unlike the proposed case of anionic Gly- and gamma-aminobutyric acid-gated ion channels, positively charged residues do not play the key role in the selection of ionic charge by the GluClbeta receptor. Taken together with measurements of the effective open pore diameter and with structural modeling, the study presented here collectively indicates that in the most constricted part of the open GluClbeta receptor's channel, Cl(-) interacts with backbone amides, where it undergoes partial dehydration necessary for traversing the pore.

Molecular tuning of fast gating in pentameric ligand-gated ion channels.

Neurotransmitters such as acetylcholine (ACh) and glycine mediate fast synaptic neurotransmission by activating pentameric ligand-gated 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 tertiary/quaternary 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 alpha7 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 alpha7 nAChR amino acid residues of the M2-M3 loop (2-3L) from the IPD control the fast activation rates of the alpha7/Gly 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.

Pore conformations and gating mechanism of a Cys-loop receptor.

Neurons regulate the propagation of chemoelectric signals throughout the nervous system by opening and closing ion channels, a process known as gating. Here, histidine-based metal-binding sites were engineered along the intrinsic pore of a chimeric Cys-loop receptor to probe state-dependent Zn(2+)-channel interactions. Patterns of Zn(2+) ion binding within the pore reveal that, in the closed state, the five pore-lining segments adopt an oblique orientation relative to the axis of ion conduction and constrict into a physical gate at their intracellular end. The interactions of Zn(2+) with the open state indicate that the five pore-lining segments should rigidly tilt to enable the movement of their intracellular ends away from the axis of ion conduction, so as to open the constriction (i.e., the gate). Alignment of the functional results with the 3D structure of an acetylcholine receptor allowed us to generate structural models accounting for the closed and open pore conformations and for a gating mechanism of a Cys-loop receptor.

Distinct subcellular targeting of fluorescent nicotinic alpha 3 beta 4 and serotoninergic 5-HT3A receptors in hippocampal neurons.

The nicotinic acetylcholine receptors (nAChRs) and the 5-HT3 serotonin receptor subtype belong to a superfamily of neurotransmitter-gated ion channels involved in fast synaptic communication throughout the nervous system. Their trafficking to the neuron plasmalemma, as well as their targeting to specific subcellular compartments, is critical for understanding their physiological role. In order to investigate the cellular distribution of these receptors, we tagged the N-termini of alpha3beta4-nAChR subunits and the 5-HT3AR subunit with cyan and yellow fluorescent proteins (CFP, YFP). The fusion subunits were coexpressed in human embryonic kidney (HEK-293) cells, where they assemble into functional receptor channels, as well as in primary cultures of hippocampal neurons. Fluorescence microscopy of living cells revealed that the heteropentameric alpha3CFP-beta4 and YFP-alpha3beta4 receptors are mainly distributed in the endoplasmic reticulum, while the homopentameric YFP-5-HT3A receptor was localized both to the plasma membrane and within intracellular compartments. Moreover, the YFP-5-HT3A receptor was found to be targeted to the micropodia in HEK-293 cells and to the dendritic spines in hippocampal neurons, where it could be accessed by extracellularly applied specific fluorescent probes. The efficient targeting of the YFP-5-HT3A to the cytoplasmic membrane is in line with the large serotonin-elicited currents (nA range) measured by whole-cell voltage-clamp recordings in transfected HEK-293 cells. In contrast, alpha3beta4-nAChRs expressed in the same cells yielded weaker ACh-evoked responses. Taken together, the fluorescent and electrophysiological studies presented here demonstrate the predominant intracellular location of alpha3beta4-nACh receptors and the predominant expression of the 5-HT3AR in dendritic surface loci.