Gamma aminobutric acid (GABA) is the major inhibitory neurotransmitter in the adult brain and mechanisms of GABAergic inhibition have been intensely investigated in the past decades. Recent studies provided evidence for an important role of astrocytes in shaping GABAergic currents. One of the most obvious, but yet poorly understood, mechanisms of the cross-talk between GABAergic currents and astrocytes is metabolism including neurotransmitter homeostasis. In particular, how modulation of GABAergic currents by astrocytes depends on key enzymes involved in cellular metabolism remains largely unknown. To address this issue, we have considered two simple models of neuronal culture (NC): nominally astrocyte-free NC and neuronal-astrocytic co-cultures (ANCC). Miniature Inhibitory Postsynaptic Currents (mIPSCs) were recorded in control conditions and in the presence of different enzyme blockers. We report that enrichment of NC with astrocytes results in a marked increase in mIPSC frequency. This enhancement of GABAergic activity was accompanied by increased number of GAD65 and vGAT puncta, indicating that at least a part of the frequency enhancement was due to increased number of synaptic contacts. Inhibition of glutamine synthetase (Glns) (with MSO) strongly reduced mIPSC frequency in ANCC but had no effect in NC. Moreover, treatment of ANCC with inhibitor of glycogen phosphorylase (Gys) (BAYU6751) or with selective inhibitor of astrocytic Krebs cycle, fluoroacetate, resulted in a marked reduction of mIPSC frequency in ANCC having no effect in NC. We conclude that GABAergic synaptic transmission strongly depends on neuron-astrocyte interaction in a manner dependent on key metabolic enzymes as well as on the Krebs cycle.

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The GABA A receptor is a member of the Cys-loop family and plays a crucial role in the adult mammalian brain inhibition. Although the static structure of this receptor is emerging, the molecular mechanisms underlying its conformational transitions remain elusive. It is known that in the Cys-loop receptors, the interface between extracellular and transmembrane domains plays a key role in transmitting the “activation wave” down to the channel gate in the pore. It has been previously reported that histidine 55 (H55), located centrally at the interfacial β1−β2 loop of the α 1 subunit, is important in the receptor activation, but it is unknown which specific gating steps it is affecting. In the present study, we addressed this issue by taking advantage of the state-of-the-art macroscopic and single-channel recordings together with extensive modeling. Considering that H55 is known to affect the local electrostatic landscape and because it is neighbored by two negatively charged aspartates, a well conserved feature in the α subunits, we considered substitution with negative (E) and positive (K) residues. We found that these mutations markedly affected the receptor gating, altering primarily preactivation and desensitization transitions. Importantly, opposite effects were observed for these two mutations strongly suggesting involvement of electrostatic interactions. Single-channel recordings suggested also a minor effect on opening/closing transitions which did not depend on the electric charge of the substituting amino acid. Altogether, we demonstrate that H55 mutations affect primarily preactivation and desensitization most likely by influencing local electrostatic interactions at the receptor interface.

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.

Cys-loop receptors are a superfamily of transmembrane, pentameric receptors that play a crucial role in mammalian CNS signaling. Physiological activation of these receptors is typically initiated by neurotransmitter binding to the orthosteric binding site, located at the extracellular domain (ECD), which leads to the opening of the channel pore (gate) at the transmembrane domain (TMD). Whereas considerable knowledge on molecular mechanisms of Cys-loop receptor activation was gathered for the acetylcholine receptor, little is known with this respect about the GABA A receptor (GABA A R), which mediates cellular inhibition. Importantly, several static structures of GABA A R were recently described, paving the way to more in-depth molecular functional studies. Moreover, it has been pointed out that the TMD-ECD interface region plays a crucial role in transduction of conformational changes from the ligand binding site to the channel gate. One of the interface structures implicated in this transduction process is the M2-M3 loop with a highly conserved proline (P277) residue. To address this issue specifically for α 1 β 2 γ 2L GABA A R, we choose to substitute proline α 1 P277 with amino acids with different physicochemical features such as electrostatic charge or their ability to change the loop flexibility. To address the functional impact of these mutations, we performed macroscopic and single-channel patch-clamp analyses together with modeling. Our findings revealed that mutation of α 1 P277 weakly affected agonist binding but was critical for all transitions of GABA A R gating: opening/closing, preactivation, and desensitization. In conclusion, we provide evidence that conservative α 1 P277 at the interface is strongly involved in regulating the receptor gating.