Search Results (6)

Although some GABA_A receptor subtypes are involved in both the passive permeability of anions and the ATP-dependent recovery of neuronal anion concentrations, the molecular mechanisms that ensure the coordination of passive and active transport processes remain unclear. Here we used fluorescence measurements to investigate the role of genistein (tyrosine kinase inhibitor) and vanadate (tyrosine phosphatase and ATPase inhibitor) in modulating GABA_AR-mediated [Cl⁻]_i/[HCO₃⁻]_i changes and ATPase activity in rat cortical neurons and HEK 293FT cells expressing the heteropentameric α2β3γ2 GABA_AR isoform. We found that genistein plays an important role in the inhibition of passive GABA_AR-mediated Cl⁻ influx and Cl⁻ATPase activity, whereas vanadate plays an important role in the inhibition of Cl⁻, HCO₃⁻ATPase activity and ATP-dependent recovery of [HCO₃⁻]_i via changes in the formation of the phosphorylated intermediate. The effect of blockers was significantly restored in the presence of phenol. In behavioral experiments, the administration of phenol has been established to induce tremors and head twitching in rats, with the involvement of GABA_AR/ATPase in these behavioral responses. Genistein can reduce the adverse effects of phenol, thereby confirming the interaction of these chemicals when binding to binding receptor sites. While our data demonstrate the opposing roles of genistein and vanadate in modulating GABA_AR/ATPase function in a bicarbonate-dependent manner. Such multidirectional systems are considered to be bistable elements involved in the regulatory mechanisms of synaptic plasticity. Full article

Unexpectedly, the affinity of the seven-transmembrane muscarinic acetylcholine receptors for their agonists is modulated by membrane depolarization. Recent reports attribute this characteristic to an embedded charge movement in the muscarinic receptor, acting as a voltage sensor. However, this explanation is inconsistent with the results of experiments measuring acetylcholine binding to muscarinic receptors in brain synaptoneurosomes. According to these results, the gating of the voltage-dependent sodium channel (VDSC) acts as the voltage sensor, generating activation of Go-proteins in response to membrane depolarization, and this modulates the affinity of muscarinic receptors for their cholinergic agonists. Full article

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 GABA_ARs, 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 GABA_AR 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 GABA_AR/ATPase function by Zn²⁺ in neonatal and adult rats. The enzymes within the scope of GABA_AR 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 β1 subunit, whereas the β3 subunit preferably possesses the Cl⁻, HCO₃⁻ATPase activity. We demonstrated that a Zn²⁺ with variable efficacy inhibits the GABA_AR 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 GABA_AR-mediated Cl⁻ and HCO₃⁻ fluxes. Finally, by site-directed mutagenesis, we identified in the M3 domain of β subunits the cysteine residue (C313) that is essential for the manifestation of Zn²⁺ potency. Full article

γ-Aminobutyric acid type A receptors (GABA_ARs) 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 GABA_AR-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 GABA_ARs 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 β3 isoform did not exhibit the desensitization of the GABA_AR-mediated Cl⁻ influx but only the resensitization. These observations lend a new physiological significance to the β3 subunit in the manifestation of GABA_AR resensitization. Full article

Although sex differences in the brain are prevalent, the knowledge about mechanisms underlying sex-related effects on normal and pathological brain functioning is rather poor. It is known that female and male brains differ in size and connectivity. Moreover, those differences are related to neuronal morphology, synaptic plasticity, and molecular signaling pathways. Among different processes assuring proper synapse functions are posttranslational modifications, and among them, S-palmitoylation (S-PALM) emerges as a crucial mechanism regulating synaptic integrity. Protein S-PALM is governed by a family of palmitoyl acyltransferases, also known as DHHC proteins. Here we focused on the sex-related functional importance of DHHC7 acyltransferase because of its S-PALM action over different synaptic proteins as well as sex steroid receptors. Using the mass spectrometry-based PANIMoni method, we identified sex-dependent differences in the S-PALM of synaptic proteins potentially involved in the regulation of membrane excitability and synaptic transmission as well as in the signaling of proteins involved in the structural plasticity of dendritic spines. To determine a mechanistic source for obtained sex-dependent changes in protein S-PALM, we analyzed synaptoneurosomes isolated from DHHC7-/- (DHHC7KO) female and male mice. Our data showed sex-dependent action of DHHC7 acyltransferase. Furthermore, we revealed that different S-PALM proteins control the same biological processes in male and female synapses. Full article

The impact of excitotoxicity mediated by N-methyl-D-aspartate (NMDA) receptor overactivation and voltage gated calcium channel (VGCC) depolarization is prominent among the postulated processes involved in the development of neurodegenerative disorders. NGP1-01, a polycyclic amine, has been shown to be neuroprotective through modulation of the NMDA receptor and VGCC, and attenuation of MPP⁺-induced neurotoxicity. Recently, we reported on the calcium modulating effects of tricycloundecene derivatives, structurally similar to NGP1-01, on the NMDA receptor and VGCC of synaptoneurosomes. In the present study, we investigated novel 4-oxatricyclo[5.2.1.0^2,6]dec-8-ene-3,5-dione derivatives for their cytotoxicity, neuroprotective effects via attenuation of MPP⁺-induced neurotoxicity and calcium influx inhibition abilities through the NMDA receptor and VGCC using neuroblastoma SH-SY5Y cells. All compounds, in general, showed low or no toxicity against neuroblastoma cells at 10–50 µM concentrations. At 10 µM, all compounds significantly attenuated MPP⁺-induced neurotoxicity as evident by the enhancement in cell viability between 23.05 ± 3.45% to 53.56 ± 9.29%. In comparison to known active compounds, the derivatives demonstrated mono or dual calcium modulating effect on the NMDA receptor and/or VGCC. Molecular docking studies using the NMDA receptor protein structure indicated that the compounds are able to bind in a comparable manner to the crystallographic pose of MK-801 inside the NMDA ion channel. The biological characteristics, together with results from in silico studies, suggest that these compounds could act as neuroprotective agents for the purpose of halting or slowing down the degenerative processes in neuronal cells. Full article