Molecular Requirements for Ethanol Differential Allosteric Modulation of Ligand-Gated Ion Channels Based on Selective G Beta Gamma Modulation

It is now believed that the allosteric modulation produced by ethanol in glycine receptors (GlyRs) depends on alcohol binding to discrete sites within the protein structure. Thus, the differential ethanol sensitivity of diverse GlyR isoforms and mutants was explained by the presence of specific residues in putative alcohol pockets. Here, we demonstrate that ethanol sensitivity in two LGIC members, the GlyR adult alpha1 and embryonic alpha2 subunits, can be modified through selective mutations that rescued or impaired Gbetagamma modulation. Even though that both isoforms were able to physically interact with Gbetagamma, only the alpha1 GlyR was functionally modulated by Gbetagamma and pharmacological ethanol concentrations. Remarkably, the simultaneous switching of two transmembrane and a single extracellular residue in alpha2 GlyRs was enough to generate GlyRs modulated by Gbetagamma and low ethanol concentrations. Interestingly, while we found that these TM residues were different to those in the alcohol binding site, the extracellular residue was recently implicated in conformational changes important to generate a pre-open activated state that precedes ion channel gating. Thus, these results support the idea that the differential ethanol sensitivity of these two GlyR isoforms rests on conformational changes in transmembrane and extracellular residues within the ion channel structure rather than in differences in alcohol binding pockets. Our results describe the molecular basis for the differential ethanol sensitivity of two LGIC members based on selective Gbetagamma modulation and provide a new mechanistic framework for allosteric modulations of abuse drugs

Conformational variability of the glycine receptor M2 domain in response to activation by different agonists

Models describing the structural changes mediating cys-loop receptor activation generally give little attention to the possibility that different agonists may promote activation via distinct M2 pore-lining domain structural rearrangements. We investigated this question by comparing the effects of different ligands on the conformation of the external portion of the homomeric α1 glycine receptor M2 domain. Conformational flexibility was assessed by tethering a rhodamine fluorophore to cysteines introduced at the 19’ or 22’ positions and monitoring fluorescence and current changes during channel activation. During glycine activation, fluorescence of the label attached to R19’C increased by ~20% and the emission peak shifted to lower wavelengths, consistent with a more hydrophobic fluorophore environment. In contrast, ivermectin activated the receptors without producing a fluorescence change. Although taurine and β-alanine were weak partial agonists at the a1R19’C GlyR, they induced large fluorescence changes. Propofol, which drastically enhanced these currents, did not induce a glycine-like blue-shift in the spectral emission peak. The inhibitors, strychnine and picrotoxin, elicited fluorescence and current changes as expected for a competitive antagonist and an open channel blocker, respectively. Glycine and taurine (or β-alanine) also produced an increase and a decrease, respectively, in the fluorescence of a label attached to the nearby L22’C residue. Thus, results from two separate labelled residues support the conclusion that the GlyR M2 domain responds with distinct conformational changes to activation by different agonists

Development of GABAergic and glycinergic transmission in the neonatal rat dorsal horn

Cutaneous spinal sensory transmission appears to lack inhibitory control in the newborn spinal cord, but the properties of GABAergic and glycinergic synapses in the neonatal dorsal horn have not been characterized. Whole-cell patch-clamp recordings from rat superficial dorsal horn neurons in spinal cord slices at postnatal day 0 (P0) to P2, P6 - P7, and P13 - P14 revealed an age-dependent increase in the frequency of spontaneous IPSCs, which were abolished by the GABA(A) receptor (GABA(A)R) antagonist bicuculline between P0 and P7 but not at P14. GABA(A)R-mediated miniature IPSCs (mIPSCs), but not glycinergic mIPSCs, were present at birth, and GABA mIPSCs remained more frequent than glycine mIPSCs at all ages. Sciatic nerve stimulation resulted in IPSCs with both GABAergic and glycinergic components, although a larger contribution arose from GABAA receptors at all ages. In gramicidin perforated patch-clamp recordings, exogenous GABA applications produced depolarization in 40% of neurons at P0 - P2, but the reversal potential of GABA-evoked currents (E-GABA) was consistently more negative than action potential threshold at this age. By P6 - P7, GABA evoked only membrane hyperpolarization. The GABA(B)R agonist baclofen elicited an outward current in all neurons with peak amplitudes observed by P6 - P7 and abolished sciatic nerve-evoked monosynaptic glutamatergic EPSCs in all groups. The results show considerable postnatal development of inhibitory processing in the dorsal horn with GABAergic mechanisms initially dominant over glycinergic events. GABA(A)R-mediated depolarizations during the first postnatal week are likely to be important for the maturation of spinal networks but do not provide a major excitatory drive to the newborn dorsal horn

Neurotransmitter-gated ion channels at fast chemical synapses: from structure to pathology

Fil: Bouzat, Cecilia Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentin

A Cation-π Interaction in the Binding Site of the Glycine Receptor Is Mediated by a Phenylalanine Residue

Cys-loop receptor binding sites characteristically contain many aromatic amino acids. In nicotinic ACh and 5-HT3 receptors, a Trp residue forms a cation-{pi} interaction with the agonist, whereas in GABAA receptors, a Tyr performs this role. The glycine receptor binding site, however, contains predominantly Phe residues. Homology models suggest that two of these Phe side chains, Phe159 and Phe207, and possibly a third, Phe63, are positioned such that they could contribute to a cation-{pi} interaction with the primary amine of glycine. Here, we test this hypothesis by incorporation of a series of fluorinated Phe derivatives using unnatural amino acid mutagenesis. The data reveal a clear correlation between the glycine EC50 value and the cation-{pi} binding ability of the fluorinated Phe derivatives at position 159, but not at positions 207 or 63, indicating a single cation-{pi} interaction between glycine and Phe159. The data thus provide an anchor point for locating glycine in its binding site, and demonstrate for the first time a cation-{pi} interaction between Phe and a neurotransmitter

The High-z Quasar Hubble Diagram

Two recent discoveries have made it possible for us to begin using high-z quasars as standard candles to construct a Hubble Diagram (HD) at z > 6. These are (1) the recognition from reverberation mapping that a relationship exists between the optical/UV luminosity and the distance of line-emitting gas from the central ionizing source. Thus, together with a measurement of the velocity of the line-emitting gas, e.g., via the width of BLR lines, such as Mg II, a single observation can therefore in principle provide a determination of the black hole's mass; and (2) the identification of quasar ULAS J1120+0641 at z = 7.085, which has significantly extended the redshift range of these sources, providing essential leverage when fitting theoretical luminosity distances to the data. In this paper, we use the observed fluxes and Mg II line-widths of these sources to show that one may reasonably test the predicted high-z distance versus redshift relationship, and we assemble a sample of 20 currently available high-z quasars for this exercise. We find a good match between theory and observations, suggesting that a more complete, high-quality survey may indeed eventually produce an HD to complement the highly-detailed study already underway (e.g., with Type Ia SNe, GRBs, and cosmic chronometers) at lower redshifts. With the modest sample we have here, we show that the R_h=ct Universe and LCDM both fit the data quite well, though the smaller number of free parameters in the former produces a more favorable outcome when we calculate likelihoods using the Akaike, Kullback, and Bayes Information Criteria. These three statistical tools result in similar probabilities, indicating that the R_h=ct Universe is more likely than LCDM to be correct, by a ratio of about 85% to 15%.Comment: Accepted for publication in JCA

Openings of the rat recombinant alpha1 homomeric glycine receptor as a function of the number of sgonist molecules bound

The functional properties of rat homomeric {alpha}1 glycine receptors were investigated using whole-cell and outside-out recording from human embryonic kidney cells transfected with rat {alpha}1 subunit cDNA. Whole-cell dose-response curves gave EC50 estimates between 30 and 120 µM and a Hill slope of ~3.3. Single channel recordings were obtained by steady-state application of glycine (0.3, 1, or 10 µM) to outside-out patches. Single channel conductances were mostly 60–90 pS, but smaller conductances of ~40 pS were also seen (10% of the events) with a relative frequency that did not depend on agonist concentration. The time constants of the apparent open time distributions did not vary with agonist concentration, but short events were more frequent at low glycine concentrations. There was also evidence of a previously missed short-lived open state that was more common at lower glycine concentrations. The time constants for the different components of the burst length distributions were found to have similar values at different concentrations. Nevertheless, the mean burst length increased with increasing glycine. This was because the relative area of each burst-length component was concentration dependent and short bursts were favored at lower glycine concentrations. Durations of adjacent open and shut times were found to be strongly (negatively) correlated. Additionally, long bursts were made up of longer than average openings separated by short gaps, whereas short bursts usually consisted of single isolated short openings. The most plausible explanation for these findings is that long bursts are generated when a higher proportion of the five potential agonist binding sites on the receptor is occupied by glycine. On the basis of the concentration dependence and the intraburst structure we provide a preliminary kinetic scheme for the activation of the homomeric glycine receptor, in which any number of glycine molecules from one to five can open the channel, although not with equal efficiency

Novel ideas about emergent vacua

Arguments for special emergent vacua which generate fermion and weak boson masses are outlined. Limitations and consequences of the concept are discussed. If confirmed the Australian dipole would give strong support to such a picture. Preliminary support from recent DZero and CDF data is discussed and predictions for LHC are presented.Comment: 8 pages, 4 figures, The interpretation of new experimental results is expanded to include the preliminary CDF boso

A comparison of glycine-and ivermectin-mediated conformational changes in the glycine receptor ligand-binding domain

Glycine receptor chloride channels are Cys-loop receptor proteins that isomerize between a low affinity closed state and a high affinity ion-conducting state. There is currently much interest in understanding the mechanisms that link affinity changes with conductance changes. This essentially involves an agonist binding in the glycine receptor ligand-binding site initiating local conformational changes that propagate in a wave towards the channel gate. However, it has proved difficult to convincingly distinguish those agonist-induced domain movements that are critical for triggering activation from those that are simply local deformations to accommodate ligands in the site. We employed voltage-clamp fluorometry to compare conformational changes in the ligand-binding site in response to activation by glycine, which binds locally, and ivermectin, which binds in the transmembrane domain. We reasoned that ivermectin-mediated activation should initiate a conformational wave that propagates from the pore-lining domain towards the ligand-binding domain, eliciting conformational changes in those extracellular domains that are allosterically linked to the gate. We found that ivermectin indeed elicited conformational changes in ligand-binding domain loops C, D and F. This implies that conformational changes in these domains are important for activation. This result also provides a mechanism to explain how ivermectin potentiates glycine-induced channel activation