Phenols and GABAA receptors: from structure and molecular mechanisms action to neuropsychiatric sequelae.

γ-Aminobutyric acid type A receptors (GABAARs) are members of the pentameric ligand-gated ion channel (pLGIC) family, which are widespread throughout the invertebrate and vertebrate central nervous system. GABAARs are engaged in short-term changes of the neuronal concentrations of chloride (Cl-) and bicarbonate (HCO3 -) ions by their passive permeability through the ion channel pore. GABAARs are regulated by various structurally diverse phenolic substances ranging from simple phenols to complex polyphenols. The wide chemical and structural variability of phenols suggest similar and different binding sites on GABAARs, allowing them to manifest themselves as activators, inhibitors, or allosteric ligands of GABAAR function. Interest in phenols is associated with their great potential for GABAAR modulation, but also with their subsequent negative or positive role in neurological and psychiatric disorders. This review focuses on the GABAergic deficit hypotheses during neurological and psychiatric disorders induced by various phenols. We summarize the structure-activity relationship of general phenol groups concerning their differential roles in the manifestation of neuropsychiatric symptoms. We describe and analyze the role of GABAAR subunits in manifesting various neuropathologies and the molecular mechanisms underlying their modulation by phenols. Finally, we discuss how phenol drugs can modulate GABAAR activity via desensitization and resensitization. We also demonstrate a novel pharmacological approach to treat neuropsychiatric disorders via regulation of receptor phosphorylation/dephosphorylation.

Zinc Inhibits the GABAAR/ATPase during Postnatal Rat Development: The Role of Cysteine Residue.

Zinc ions (Zn2+) are concentrated in various brain regions and can act as a neuromodulator, targeting a wide spectrum of postsynaptic receptors and enzymes. Zn2+ inhibits the GABAARs, and its potency is profoundly affected by the subunit composition and neuronal developmental stage. Although the extracellular amino acid residues of the receptor's hetero-oligomeric structure are preferred for Zn2+ binding, there are intracellular sites that, in principle, could coordinate its potency. However, their role in modulating the receptor function during postembryonic development remains unclear. The GABAAR possesses an intracellular ATPase that enables the energy-dependent anion transport via a pore. Here, we propose a mechanistic and molecular basis for the inhibition of intracellular GABAAR/ATPase function by Zn2+ in neonatal and adult rats. The enzymes within the scope of GABAAR performance as Cl-ATPase and then as Cl-, HCO3-ATPase form during the first week of postnatal rat development. In addition, we have shown that the Cl-ATPase form belongs to the ß1 subunit, whereas the ß3 subunit preferably possesses the Cl-, HCO3-ATPase activity. We demonstrated that a Zn2+ with variable efficacy inhibits the GABAAR as well as the ATPase activities of immature or mature neurons. Using fluorescence recording in the cortical synaptoneurosomes (SNs), we showed a competitive association between Zn2+ and NEM in parallel changes both in the ATPase activity and the GABAAR-mediated Cl- and HCO3- fluxes. Finally, by site-directed mutagenesis, we identified in the M3 domain of ß subunits the cysteine residue (C313) that is essential for the manifestation of Zn2+ potency.

Physiological Role of ATPase for GABAA Receptor Resensitization.

γ-Aminobutyric acid type A receptors (GABAARs) mediate primarily inhibitory synaptic transmission in the central nervous system. Following fast-paced activation, which provides the selective flow of mainly chloride (Cl-) and less bicarbonate (HCO3-) ions via the pore, these receptors undergo desensitization that is paradoxically prevented by the process of their recovery, referred to as resensitization. To clarify the mechanism of resensitization, we used the cortical synaptoneurosomes from the rat brain and HEK 293FT cells. Here, we describe the effect of γ-phosphate analogues (γPAs) that mimic various states of ATP hydrolysis on GABAAR-mediated Cl- and HCO3- fluxes in response to the first and repeated application of the agonist. We found that depending on the presence of bicarbonate, opened and desensitized states of the wild or chimeric GABAARs had different sensitivities to γPAs. This study presents the evidence that recovery of neuronal Cl- and HCO3- concentrations after desensitization is accompanied by a change in the intracellular ATP concentration via ATPase performance. The transition between the desensitization and resensitization states was linked to changes in both conformation and phosphorylation. In addition, the chimeric ß3 isoform did not exhibit the desensitization of the GABAAR-mediated Cl- influx but only the resensitization. These observations lend a new physiological significance to the ß3 subunit in the manifestation of GABAAR resensitization.

Ectopic GABAA receptor ß3 subunit determines Cl- / HCO3- -ATPase and chloride transport in HEK 293FT cells.

Neuronal intracellular chloride concentration ([Cl- ]i ) is a crucial determinant of transmission mediated by the γ-aminobutyric acid type A receptor (GABAA R), which subserves synaptic and extrasynaptic inhibition as well as excitation. The Cl- ion is the main carrier of charge through the GABAA R; however, bicarbonate ions ( HCO3- ) flowing in the opposite direction can also contribute to the net current. The direction of Cl- and HCO3- fluxes is determined by the underlying electrochemical gradient, which is controlled by Cl- transporters and channels. Accumulating evidence suggests that active mechanisms of chloride transport across the GABAA R pore can underlie the regulation of [Cl- ]i . Measurement of Cl- / HCO3- -ATPase activity and Cl- transport in HEK 293FT cells expressing homomeric or heteromeric GABAA R ensembles (α2, ß3, or γ2) with fluorescent dye for chloride demonstrated that receptor subtypes containing the ß3 subunit show enzymatic activity and participate in GABA-mediated or ATP-dependent Cl- transport. GABA-mediated flow of Cl- ions into and out of the cells occurred for a short time period but then rapidly declined. However, Cl- ion flux was stabilized for a long time period in the presence of HCO3- ions. The reconstituted ß3 subunit isoform, purified as a fusion protein, confirmed that ß3 is critical for ATPase; however, only the triplet variant showed the full receptor function. The high sensitivity of the enzyme to γ-phosphate inhibitors led us to postulate that the ß3 subunit is catalytic. Our discovery of a GABAA R type that requires ATP consumption for chloride movement provides new insight into the molecular mechanisms of inhibitory signaling.

Transcriptomic changes triggered by ouabain in rat cerebellum granule cells: Role of α3- and α1-Na+,K+-ATPase-mediated signaling.

It was shown previously that inhibition of the ubiquitous α1 isoform of Na+,K+-ATPase by ouabain sharply affects gene expression profile via elevation of intracellular [Na+]i/[K+]i ratio. Unlike other cells, neurons are abundant in the α3 isoform of Na+,K+-ATPase, whose affinity in rodents to ouabain is 104-fold higher compared to the α1 isoform. With these sharp differences in mind, we compared transcriptomic changes in rat cerebellum granule cells triggered by inhibition of α1- and α3-Na+,K+-ATPase isoforms. Inhibition of α1- and α3-Na+,K+-ATPase isoforms by 1 mM ouabain resulted in dissipation of transmembrane Na+ and K+ gradients and differential expression of 994 transcripts, whereas selective inhibition of α3-Na+,K+-ATPase isoform by 100 nM ouabain affected expression of 144 transcripts without any impact on the [Na+]i/[K+]i ratio. The list of genes whose expression was affected by 1 mM ouabain by more than 2-fold was abundant in intermediates of intracellular signaling and transcription regulators, including augmented content of Npas4, Fos, Junb, Atf3, and Klf4 mRNAs, whose upregulated expression was demonstrated in neurons subjected to electrical and glutamatergic stimulation. The role [Na+]i/[K+]i-mediated signaling in transcriptomic changes involved in memory formation and storage should be examined further.

Differential processing of small RNAs during endoplasmic reticulum stress.

The accumulation of misfolded proteins in the endoplasmic reticulum (ER) lumen due to the disruption of the homeostatic system of the ER leads to the induction of the ER stress response. Cellular stress-induced pathways globally transform genes expression on both the transcriptional and post-transcriptional levels with small RNA involvement as regulators of the stress response. The modulation of small RNA processing might represent an additional layer of a complex stress response program. However, it is poorly understood. Here, we studied changes in expression and small RNAs processing upon ER stress in Jurkat T-cells. Induced by ER-stress, depletion of miRNAs among small RNA composition was accompanied by a global decrease of 3' mono-adenylated, mono-cytodinylated and a global increase of 3' mono-uridinylated miRNA isoforms. We observed the specific subset of differentially expressed microRNAs, and also the dramatic induction of 32-nt tRNA fragments precisely phased to 5' and 3' ends of tRNA from a subset of tRNA isotypes. The induction of these tRNA fragments was linked to Angiogenin RNase, which mediates translation inhibition. Overall, the global perturbations of the expression and processing of miRNAs and tiRNAs were the most prominent features of small RNA transcriptome changes upon ER stress.