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

Presence of Inhibitory Glycinergic Transmission in Medium Spiny Neurons in the Nucleus Accumbens

It is believed that the rewarding actions of drugs are mediated by dysregulation of the mesolimbic dopaminergic system leading to increased levels of dopamine in the nucleus accumbens (nAc). It is widely recognized that GABAergic transmission is critical for neuronal inhibition within nAc. However, it is currently unknown if medium spiny neurons (MSNs) also receive inhibition by means of glycinergic synaptic inputs. We used a combination of proteomic and electrophysiology studies to characterize the presence of glycinergic input into MSNs from nAc demonstrating the presence of glycine transmission into nAc. In D1 MSNs, we found low frequency glycinergic miniature inhibitory postsynaptic currents (mIPSCs) which were blocked by 1 μM strychnine (STN), insensitive to low (10, 50 mM) and high (100 mM) ethanol (EtOH) concentrations, but sensitive to 30 μM propofol. Optogenetic experiments confirmed the existence of STN-sensitive glycinergic IPSCs and suggest a contribution of GABA and glycine neurotransmitters to the IPSCs in nAc. The study reveals the presence of glycinergic transmission in a non-spinal region and opens the possibility of a novel mechanism for the regulation of the reward pathway

A Single Phenylalanine Residue in the Main Intracellular Loop of [alpha]1 [gamma]-Aminobutyric Acid Type A and Glycine Receptors Influences Their Sensitivity to Propofol

BACKGROUND: The intravenous anaesthetic propofol acts as a positive allosteric modulator of glycine (GlyRs) and γ-aminobutyric acid type A (GABA(A)Rs) receptors. Although the role of transmembrane residues is recognized, little is known about the involvement of other regions in the modulatory effects of propofol. Therefore, we explored the influence of the large intracellular loop (LIL) in propofol sensitivity of both receptors. METHODS: We screened the LIL of α(1) GlyRs and α(1)β(2) GABA(A)Rs using alanine replacement. Sensitivity to propofol was studied using patch-clamp recording in HEK293 cells transiently transfected with WT (wild type) or mutant receptors. RESULTS: Alanine mutation of a conserved phenylalanine residue within the α(1) LIL significantly reduced propofol enhancement in both GlyRs (360±30 vs 75±10%, mean±SEM) and GABA(A)Rs (361±49% vs 80±23%). Remarkably, propofol-hyposensitive mutant receptors retained their sensitivity to other allosteric modulators such as alcohols, etomidate, trichloroethanol and isoflurane. At the single channel level, the ability of propofol to increase open probability was significantly reduced in both α(1) GlyR (189±36 vs 22±13%) and α(1)β(2) GABA(A)R (279±29 vs 29±11%) mutant receptors. CONCLUSION: In this study, we demonstrate that the LIL of both GlyR and GABA(A)R has a conserved single phenylalanine residue (F380 and F385, respectively) that influences its sensitivity to propofol. Our results suggest a new role of the LIL in the allosteric modulation of two members of the Cys-loop superfamily. Thus, these data provide new insights into the molecular framework behind the modulation of inhibitory ion channels by propofol

The Basic Property of Lys385 Is Important for Potentiation of the Human alpha1 Glycine Receptor by Ethanol.

peer reviewedEthanol alters the function of several members of the Cys-loop ligand-gated ion channel superfamily. Recent studies have shown that the sensitivity of the alpha1 glycine receptor (GlyR) to ethanol can be affected by the state of G protein activation mediated by the interaction of Gbetagamma with intracellular amino acids in the GlyR. Here, we evaluated the physicochemical property of Lys385 that contributes to ethanol modulation by using mutagenesis, patch-clamp, and biochemical techniques. A conserved substitution (K385R) did not affect either the apparent glycine EC(50) (40 +/- 1 versus 41 +/- 0.5 muM) or the ethanol-induced potentiation (53 +/- 5 versus 46 +/- 5%) of the human alpha1 GlyR. On the other hand, replacement of this residue with glutamic acid (K385E), an acidic amino acid, reduced the potentiation of the GlyR to 10 +/- 1%. Furthermore, mutations with a hydrophobic leucine (K385L), a hydrogen bond donor glutamine (K385Q), or a neutral residue (K385A) also reduced ethanol modulation. Finally, substitution by a large and hydrophobic residue (K385F) and deletion of 385 (Lys385_) reduced ethanol modulation to 10 +/- 4 and 17 +/- 0.4%, respectively. Experiments using dynamic cysteine substitution with a methanethiosulfonate reagent and homology modeling indicate that the basic property and the position of Lys385, probably because of its interaction with Gbetagamma, is critical for ethanol potentiation of the receptor

Glycine Receptors Involved in Synaptic Transmission Are Selectively Regulated By the Cytoskeleton in Mouse Spinal Neurons

Using whole cell patch-clamp recordings, we examined the effect of colchicine, a microtubule disrupter, on the properties of glycine receptors (GlyRs) in cultured spinal cord neurons. Confocal microscopy revealed that colchicine treatment effectively altered microtubule bundles and neuronal morphology. Application of colchicine via the culture media or the patch-pipette, however, did not affect the whole cell current rundown (73 ± 6% of control after 1 h), the sensitivity of the GlyR to glycine (EC50 = 29 ± 1 μM), or strychnine inhibition (47 ± 5% of control after 100 nM strychnine). On the other hand, colchicine dialyzed for 25 min via the patch pipette selectively reduced the quantal amplitude of spontaneous glycinergic miniature inhibitory postsynaptic currents (mIPSCs) to 68 ± 5% of control. This effect was specific for GlyRs since synaptic events mediated by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and GABAAreceptors were unchanged. In conclusion, this study indicates that microtubules can regulate the function of GlyRs involved in inhibitory synaptic transmission

Glycine Receptors Involved in Synaptic Transmission Are Selectively Regulated By the Cytoskeleton in Mouse Spinal Neurons

Using whole cell patch-clamp recordings, we examined the effect of colchicine, a microtubule disrupter, on the properties of glycine receptors (GlyRs) in cultured spinal cord neurons. Confocal microscopy revealed that colchicine treatment effectively altered microtubule bundles and neuronal morphology. Application of colchicine via the culture media or the patch-pipette, however, did not affect the whole cell current rundown (73 ± 6% of control after 1 h), the sensitivity of the GlyR to glycine (EC50 = 29 ± 1 μM), or strychnine inhibition (47 ± 5% of control after 100 nM strychnine). On the other hand, colchicine dialyzed for 25 min via the patch pipette selectively reduced the quantal amplitude of spontaneous glycinergic miniature inhibitory postsynaptic currents (mIPSCs) to 68 ± 5% of control. This effect was specific for GlyRs since synaptic events mediated by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and GABAAreceptors were unchanged. In conclusion, this study indicates that microtubules can regulate the function of GlyRs involved in inhibitory synaptic transmission

Molecular determinants for G protein betagamma modulation of ionotropic glycine receptors

The ligand-gated ion channel superfamily plays a critical role in neuronal excitability. the functions of glycine receptor ( GlyR) and nicotinic acetylcholine receptor are modulated by G protein beta gamma subunits. the molecular determinants for this functional modulation, however, are still unknown. Studying mutant receptors, we identified two basic amino acid motifs within the large intracellular loop of the GlyR alpha(1) subunit that are critical for binding and functional modulation by G beta gamma. Mutations within these sequences demonstrated that all of the residues detected are important for G beta gamma modulation, although both motifs are necessary for full binding. Molecular modeling predicts that these sites are alpha-helixes near transmembrane domains 3 and 4, near to the lipid bilayer and highly electropositive. Our results demonstrate for the first time the sites for G protein beta gamma subunit modulation on GlyRs and provide a new framework regarding the ligand-gated ion channel superfamily regulation by intracellular signaling

Modulatory Actions of the Glycine Receptor β Subunit on the Positive Allosteric Modulation of Ethanol in α2 Containing Receptors

Alpha1-containing glycine receptors (GlyRs) are major mediators of synaptic inhibition in the spinal cord and brain stem. Recent studies reported the presence of α2-containing GlyRs in other brain regions, such as nucleus accumbens and cerebral cortex. GlyR activation decreases neuronal excitability associated with sensorial information, motor control, and respiratory functions; all of which are significantly altered during ethanol intoxication. We evaluated the role of β GlyR subunits and of two basic amino acid residues, K389 and R390, located in the large intracellular loop (IL) of the α2 GlyR subunit, which are important for binding and functional modulation by Gβγ, the dimer of the trimeric G protein conformation, using HEK-293 transfected cells combined with patch clamp electrophysiology. We demonstrate a new modulatory role of the β subunit on ethanol sensitivity of α2 subunits. Specifically, we found a differential allosteric modulation in homomeric α2 GlyRs compared with the α2β heteromeric conformation. Indeed, while α2 was insensitive, α2β GlyRs were substantially potentiated by ethanol, GTP-γ-S, propofol, Zn2+ and trichloroethanol. Furthermore, a Gβγ scavenger (ct-GRK2) selectively attenuated the effects of ethanol on recombinant α2β GlyRs. Mutations in an α2 GlyR co-expressed with the β subunit (α2AAβ) specifically blocked ethanol sensitivity, but not propofol potentiation. These results show a selective mechanism for low ethanol concentration effects on homomeric and heteromeric conformations of α2 GlyRs and provide a new mechanism for ethanol pharmacology, which is relevant to upper brain regions where α2 GlyRs are abundantly expressed