GABAA Receptor ß2E155 Residue Located at the Agonist-Binding Site Is Involved in the Receptor Gating.

GABAA receptors (GABAARs) play a crucial role in mediating inhibition in the adult brain. In spite of progress in describing (mainly) the static structures of this receptor, the molecular mechanisms underlying its activation remain unclear. It is known that in the α1ß2γ2L receptors, the mutation of the ß2E155 residue, at the orthosteric binding site, strongly impairs the receptor activation, but the molecular and kinetic mechanisms of this effect remain elusive. Herein, we investigated the impact of the ß2E155C mutation on binding and gating of the α1ß2γ2L receptor. To this end, we combined the macroscopic and single-channel analysis, the use of different agonists [GABA and muscimol (MSC)] and flurazepam (FLU) as a modulator. As expected, the ß2E155C mutation caused a vast right shift of the dose-response (for GABA and MSC) and, additionally, dramatic changes in the time course of current responses, indicative of alterations in gating. Mutated receptors showed reduced maximum open probability and enhanced receptor spontaneous activity. Model simulations for macroscopic currents revealed that the primary effect of the mutation was the downregulation of the preactivation (flipping) rate. Experiments with MSC and FLU further confirmed a reduction in the preactivation rate. Our single-channel analysis revealed the mutation impact mainly on the second component in the shut times distributions. Based on model simulations, this finding further confirms that this mutation affects mostly the preactivation transition, supporting thus the macroscopic data. Altogether, we provide new evidence that the ß2E155 residue is involved in both binding and gating (primarily preactivation).

Matrix metalloproteinase-3 in brain physiology and neurodegeneration.

Structural and functional synapse reorganization is one of the key issues of learning and memory mechanisms. Specific proteases, called matrix metalloproteinases (MMPs), play a pivotal role during learning-related modification of neural circuits. Different types of MMPs modify the extracellular perisynaptic environment, leading to the plastic changes in the synapses. In recent years, there has been an increasing interest in the role played by matrix metalloproteinase-3 (MMP-3) in various processes occurring in the mammalian brain, both in physiological and pathological conditions. In this review, we discuss a crucial function of MMP-3 in synaptic plasticity, learning, neuronal development, as well as in neuroregeneration. We discuss the involvement of MMP-3 in synaptic long-term potentiation, which is likely to have a profound impact on experience-dependent learning. On the other hand, we also provide examples of deleterious actions of uncontrolled MMP-3 activity on the central nervous system (CNS) and its contribution to Alzheimer's and Parkinson's diseases (AD and PD). Since the molecular mechanisms controlled by MMP-3 have a profound and diverse impact on physiological and pathological brain functioning, their deep understanding may be crucial for the development of more specific methods for the treatment of neuropsychiatric diseases.

Dietary acetylenic oxylipin falcarinol differentially modulates GABAA receptors.

The dietary oxylipins falcarinol (1a) and falcarindiol (1b) trap thiols by direct nucleophilic addition to their diyne system, but despite this, only falcarinol (1a) is a reversible agonist of cannabinoid receptors, providing a rationale for comparing their activity also on other neuronal targets. Because GABAA receptors (GABAARs) are exquisitely sensitive to polyacetylenic oxylipins in terms of either potentiation (falcarindiol, 1b) or inhibition (oenanthotoxin, 2a), the activity of 1a was investigated on synaptic (α1ß2γ2L) and extrasynaptic (α1ß2δ and α1ß2) subtypes of GABAARs. Falcarinol (1a) significantly enhanced the amplitude of currents mediated by α1ß2γ2L receptors, but this effect was associated with a use-dependent block. Conversely, α1ß2 receptors were inhibited without any sign of use-dependent block for the entire range of concentrations tested (1-10 µM). Interestingly, responses mediated by α1ß2δ receptors, showing no or very little macroscopic desensitization, were strongly potentiated by 1a, exhibiting a fading reminiscent of macroscopic desensitization. When compared to the activity of falcarindiol (1b), falcarinol (1a) showed a higher affinity for GABAARs and, overall, a substantially different profile of pharmacological action. Taken together, the present data support the view that modulation of GABAARs might underlie the insecticidal and sedative activity of falcarinol (1a).

Falcarindiol allosterically modulates GABAergic currents in cultured rat hippocampal neurons.

Falcarindiol (1), a C-17 polyacetylenic diol, shows a pleiotropic profile of bioactivity, but the mechanism(s) underlying its actions are largely unknown. Large amounts of 1 co-occur in water hemlock (Oenanthe crocata) along with the convulsant polyacetylenic toxin oenanthotoxin (2), a potent GABA(A) receptor (GABA(A)R) inhibitor. Since these compounds are structurally and biogenetically related, it was considered of interest to evaluate whether 1 could affect GABAergic activity, and for this purpose a model of hippocampal cultured neurons was used. Compound 1 significantly increased the amplitude of miniature inhibitory postsynaptic currents, accelerated their onset, and prolonged the decay kinetics. This compound enhanced also the amplitude of currents elicited by 3 µM GABA and accelerated their fading, reducing, however, currents evoked by a saturating (10 mM) GABA concentration. Moreover, kinetic analysis of responses to 10 mM GABA revealed that 1 upregulated the rate and extent of desensitization and slowed the current onset and deactivation. Taken together, these data show that 1 exerts a potent modulatory action on GABA(A)Rs, possibly by modulating agonist binding and desensitization, overall potentially decreasing the toxicity of co-occurring GABA-inhibiting convulsant toxins.

Modulation of GABAergic synaptic currents and current responses by α-thujone and dihydroumbellulone.

α-Thujone (1a), a constituent of wormwood, has been suspected to cause adverse psychoactive reactions in addicted drinkers of absinthe. While the content of 1a in absinthe is too low for such effects, at higher doses it can indeed induce seizures and inhibit GABA(A) receptors (GABA(A)Rs). The effect of 1a on GABAergic synaptic currents and the mechanisms by which it modulates GABA(A)Rs remain unknown. To address these issues, cultured hippocampal neurons were used to investigate the action of 1a on GABAergic miniature inhibitory postsynaptic currents (mIPSCs) and on responses to exogenous GABA applications. Since lipophilic compounds often show nonspecific actions related to their hydrophobicity, the action of 1a was compared to that of dihydroumbellulone (2), a configurationally pseudoenantiomeric constitutional isomer. α-Thujone (1a) reduced mIPSC frequency and amplitude and also moderately affected their kinetics, indicating both pre- and postsynaptic mechanisms. Analysis of current responses to exogenous GABA revealed that 1a reduced their amplitude, affecting their onset, desensitization, and deactivation, suggesting an effect on receptor gating. In contrast, 2 caused only a weak or negligible effect on GABAergic currents, supporting the effects of 1a on GABAergic inhibition as being due to specific interactions with GABA(A)Rs.