Bidirectional plasticity of GABAergic tonic inhibition in hippocampal somatostatin- and parvalbumin-containing interneurons.

GABAA receptors present in extrasynaptic areas mediate tonic inhibition in hippocampal neurons regulating the performance of neural networks. In this study, we investigated the effect of NMDA-induced plasticity on tonic inhibition in somatostatin- and parvalbumin-containing interneurons. Using pharmacological methods and transgenic mice (SST-Cre/PV-Cre x Ai14), we induced the plasticity of GABAergic transmission in somatostatin- and parvalbumin-containing interneurons by a brief (3 min) application of NMDA. In the whole-cell patch-clamp configuration, we measured tonic currents enhanced by specific agonists (etomidate or gaboxadol). Furthermore, in both the control and NMDA-treated groups, we examined to what extent these changes depend on the regulation of distinct subtypes of GABAA receptors. Tonic conductance in the somatostatin-containing (SST+) interneurons is enhanced after NMDA application, and the observed effect is associated with an increased content of α5-containing GABAARs. Both fast-spiking and non-fast-spiking parvalbumin-positive (PV+) cells showed a reduction of tonic inhibition after plasticity induction. This effect was accompanied in both PV+ interneuron types by a strongly reduced proportion of δ-subunit-containing GABAARs and a relatively small increase in currents mediated by α5-containing GABAARs. Both somatostatin- and parvalbumin-containing interneurons show cell type-dependent and opposite sign plasticity of tonic inhibition. The underlying mechanisms depend on the cell-specific balance of plastic changes in the contents of α5 and δ subunit-containing GABAARs.

Mutation of valine 53 at the interface between extracellular and transmembrane domains of the ß2 principal subunit affects the GABAA receptor gating.

GABAA receptors (gamma-aminobutyric acid type A receptors) are pentameric ligand-gated ion channels mediating inhibition in adult mammalian brains. Their static structure has been intensely studied in the past years but the underlying molecular activatory mechanisms remain obscure. The interface between extracellular and transmembrane domains has been recognized as a key player in the receptor gating. However, the role of the valine 53 in the ß1-ß2 loop of the principal subunit (ß2) remains controversial showing differences compared to homologous residues in some cys-loop counterparts such as nAChR. To address the role of the ß2V53 residue in the α1ß2γ2L receptor gating, we performed high resolution macroscopic and single-channel recordings. To explore underlying molecular mechanisms a variety of substituting amino acids were investigated: Glutamate and Lysine (different electric charge), Alanine (aliphatic, larger than Valine) and Histidine (same residue as in homologous α1H55). We report that mutation of the ß2V53 residue results in alterations of nearly all gating transitions including opening/closing, preactivation and desensitization. A dramatic gating impairment was observed for glutamate substitution (ß2V53E) but ß2V53K mutation had a weak effect. The impact of histidine substitution was also small while ß2V53A markedly affected the receptor but to a smaller extent than ß2V53E. Considering available structures in desensitized and bicuculline blocked shut states we propose that strongly detrimental effect of ß2V53E mutation on receptor activation results from electrostatic interaction between the glutamate and ß2K274 on the loop M2-M3 which stabilizes the receptor in the shut state. We conclude that ß2V53 is strongly involved in mechanisms underlying the receptor gating.

GABAergic synapses onto SST and PV interneurons in the CA1 hippocampal region show cell-specific and integrin-dependent plasticity.

It is known that GABAergic transmission onto pyramidal neurons shows different forms of plasticity. However, GABAergic cells innervate also other inhibitory interneurons and plasticity phenomena at these projections remain largely unknown. Several mechanisms underlying plastic changes, both at inhibitory and excitatory synapses, show dependence on integrins, key proteins mediating interaction between intra- and extracellular environment. We thus used hippocampal slices to address the impact of integrins on long-term plasticity of GABAergic synapses on specific inhibitory interneurons (containing parvalbumin, PV + or somatostatin, SST +) known to innervate distinct parts of principal cells. Administration of RGD sequence-containing peptide induced inhibitory long-term potentiation (iLTP) at fast-spiking (FS) PV + as well as on SST + interneurons. Interestingly, treatment with a more specific peptide GA(C)RRETAWA(C)GA (RRETAWA), affecting α5ß1 integrins, resulted in iLTP in SST + and iLTD in FS PV + interneurons. Brief exposure to NMDA is known to induce iLTP at GABAergic synapses on pyramidal cells. Intriguingly, application of this protocol for considered interneurons evoked iLTP in SST + and iLTD in PV + interneurons. Moreover, we showed that in SST + cells, NMDA-evoked iLTP depends on the incorporation of GABAA receptors containing α5 subunit to the synapses, and this iLTP is occluded by RRETAWA peptide, indicating a key role of α5ß1 integrins. Altogether, our results revealed that plasticity of inhibitory synapses at GABAergic cells shows interneuron-specificity and show differences in the underlying integrin-dependent mechanisms. This is the first evidence that neuronal disinhibition may be a highly plastic process depending on interneuron type and integrins' activity.

α1 Proline 277 Residues Regulate GABAAR Gating through M2-M3 Loop Interaction in the Interface Region.

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 GABAA receptor (GABAAR), which mediates cellular inhibition. Importantly, several static structures of GABAAR 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 α1ß2γ2L GABAAR, we choose to substitute proline α1P277 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 α1P277 weakly affected agonist binding but was critical for all transitions of GABAAR gating: opening/closing, preactivation, and desensitization. In conclusion, we provide evidence that conservative α1P277 at the interface is strongly involved in regulating the receptor gating.

GABAA receptor proline 273 at the interdomain interface of the ß2 subunit regulates entry into desensitization and opening/closing transitions.

AIMS: GABAA receptors belong to Cys-loop ion channel family and mediate inhibition in the brain. Despite the abundance of structural data on receptor structure, the molecular scenarios of activation are unknown. In this study we investigated the role of a ß2P273 residue in channel gating transitions. This residue is located in a central position of the M2-M3 linker of the interdomain interface, expected to be predisposed to interact with another interfacial element, the ß1-ß2 loop of the extracellular side. The interactions occurring on this interface have been reported to couple agonist binding to channel gating. MAIN METHODS: We recorded micro- and macroscopic current responses of recombinant GABAA receptors mutated at the ß2P273 residue (to A, K, E) to saturating GABA. Electrophysiological data served as basis to kinetic modeling, used to decipher which gating transition were affected by mutations. KEY FINDINGS: Mutations of this residue impaired macroscopic desensitization and accelerated current deactivation with P273E mutant showing greatest deviation from wild-type. Single-channel analysis revealed alterations mainly in short-lived shut times and shortening of openings, resulting in dramatic changes in intraburst open probability. Kinetic modeling indicated that ß2P273 mutants show diminished entry into desensitized and open states as well as faster channel closing transitions. SIGNIFICANCE: In conclusion, we demonstrate that ß2P273 of the M2-M3 linker is a crucial element of the ECD-TMD interface regulating the receptor's ability to undergo late gating transitions. Henceforth, this region could be an important target for new pharmacological tools affecting GABAAR-mediated inhibition.

Integrins Bidirectionally Regulate the Efficacy of Inhibitory Synaptic Transmission and Control GABAergic Plasticity.

For many decades, synaptic plasticity was believed to be restricted to excitatory transmission. However, in recent years, this view started to change, and now it is recognized that GABAergic synapses show distinct forms of activity-dependent long-term plasticity, but the underlying mechanisms remain obscure. Herein, we asked whether signaling mediated by ß1 or ß3 subunit-containing integrins might be involved in regulating the efficacy of GABAergic synapses, including the NMDA receptor-dependent inhibitory long-term potentiation (iLTP) in the hippocampus. We found that activation of ß3 integrin with fibrinogen induced a stable depression, whereas inhibition of ß1 integrin potentiated GABAergic synapses at CA1 pyramidal neurons in male mice. Additionally, compounds that interfere with the interaction of ß1 or ß3 integrins with extracellular matrix blocked the induction of NMDA-iLTP. In conclusion, we provide the first evidence that integrins are key players in regulating the endogenous modulatory mechanisms of GABAergic inhibition and plasticity in the hippocampus.SIGNIFICANCE STATEMENT Epilepsy, schizophrenia, and anxiety are just a few medical conditions associated with dysfunctional inhibitory synaptic transmission. GABAergic synapses are known for their extraordinary susceptibility to modulation by endogenous factors and exogenous pharmacological agents. We describe here that integrins, adhesion proteins, play a key role in the modulation of inhibitory synaptic transmission. Specifically, we show that interference with integrin-dependent adhesion results in a variety of effects on the amplitude and frequency of GABAergic mIPSCs. Activation of ß3 subunit-containing integrins induces inhibitory long-term depression, whereas the inhibition of ß1 subunit-containing integrins induces iLTP. Our results unveil an important mechanism controlling synaptic inhibition, which opens new avenues into the usage of integrin-aimed pharmaceuticals as modulators of GABAergic synapses.

Extracellular Metalloproteinases in the Plasticity of Excitatory and Inhibitory Synapses.

Long-term synaptic plasticity is shaped by the controlled reorganization of the synaptic proteome. A key component of this process is local proteolysis performed by the family of extracellular matrix metalloproteinases (MMPs). In recent years, considerable progress was achieved in identifying extracellular proteases involved in neuroplasticity phenomena and their protein substrates. Perisynaptic metalloproteinases regulate plastic changes at synapses through the processing of extracellular and membrane proteins. MMP9 was found to play a crucial role in excitatory synapses by controlling the NMDA-dependent LTP component. In addition, MMP3 regulates the L-type calcium channel-dependent form of LTP as well as the plasticity of neuronal excitability. Both MMP9 and MMP3 were implicated in memory and learning. Moreover, altered expression or mutations of different MMPs are associated with learning deficits and psychiatric disorders, including schizophrenia, addiction, or stress response. Contrary to excitatory drive, the investigation into the role of extracellular proteolysis in inhibitory synapses is only just beginning. Herein, we review the principal mechanisms of MMP involvement in the plasticity of excitatory transmission and the recently discovered role of proteolysis in inhibitory synapses. We discuss how different matrix metalloproteinases shape dynamics and turnover of synaptic adhesome and signal transduction pathways in neurons. Finally, we discuss future challenges in exploring synapse- and plasticity-specific functions of different metalloproteinases.

The ß2 subunit E155 residue as a proton sensor at the binding site on GABA type A receptors.

GABA type A receptor plays a key role in inhibitory signaling in the adult central nervous system. This receptor can be modulated by protons but the underlying molecular mechanisms have not been fully explored. To find possible pH-sensor residues, a comparative study for proton-activated GLIC channel and α1ß2γ2 GABA receptor was performed and pK 's of respective residues were estimated by numerical algorithms which consider local interactions. ß E155, located at the GABA binding site, showed pKa values close to physiological values and dependence on the receptor state and ligation, suggesting a role in modulation by pH. To validate this prediction, pH sensitivity of current responses to GABA was investigated using patch-clamp technique for WT and mutated (ß2E155[C, S, Q, L]) GABA receptors. Cysteine mutation preserved pH sensitivity. However, for remaining mutants, the sensitivity to acidification (pH = 6.0) was reduced becoming not statistically significant. The effect of alkaline pH (8.0) was maintained for all mutants with exception for ß2E155L for which it was nearly abolished. To further explore the impact of considered mutations, molecular docking was performed which indicated that pH modulation is probably affected by interplay between binding site residues, zwitterion GABA and protons. These data, altogether, indicate that mutation of ß2E155 to hydrophobic residue (L) maximally impaired pH modulation while for polar substitutions the effect was smaller. In conclusion, our data provide evidence that a key binding site residue ß2E155 plays an important role in proton sensitivity of GABA receptor.

Glycine substitution of α1F64 residue at the loop D of GABAA receptor impairs gating - Implications for importance of binding site-channel gate linker rigidity.

GABAA receptors (GABAARs) play a crucial role in mediating inhibition in adult mammalian brains. In the recent years, an impressive progress in revealing the static structure of GABAARs was achieved but the molecular mechanisms underlying their conformational transitions remain elusive. Phenylalanine 64 (α1F64) is located at the loop D of the orthosteric binding site of GABAAR and was found to directly interact with GABA molecule. Mutations of α1F64 were demonstrated to affect not only binding but also some gating properties. Loop D is a rigid ß strand which seems to be particularly suitable to convey activatory signaling from the ligand binding site (LBS) to the gate at the channel pore. To test this scenario, we have investigated the substitution of α1F64 with glycine, the smallest amino acid, widely recognized as a rigidity "reducer" of protein structures. To this end, we assessed the impact of the α1F64G mutation in the α1ß2γ2L type of GABAARs on gating properties by analyzing both macroscopic responses to rapid agonist applications and single-channel currents. We found that this substitution dramatically altered all gating features of the receptor (opening/closing, preactivation and desensitization) which contrasts with markedly weaker effects of previously considered substitutions (α1F64L and α1F64A). In particular, α1F64G mutation practically abolished the desensitization process. At the same time, the α1F64G mutant maintained gating integrity manifested as single-channel activity in the form of clusters. We conclude that rigidity of the loop D plays a crucial role in conveying the activation signal from the LBS to the channel gate.

Mutations at the M2 and M3 Transmembrane Helices of the GABAARs α1 and ß2 Subunits Affect Primarily Late Gating Transitions Including Opening/Closing and Desensitization.

GABA type A receptors (GABAARs) belong to the pentameric ligand-gated ion channel (pLGIC) family and play a crucial role in mediating inhibition in the adult mammalian brain. Recently, a major progress in determining the static structure of GABAARs was achieved, although precise molecular scenarios underlying conformational transitions remain unclear. The ligand binding sites (LBSs) are located at the extracellular domain (ECD), very distant from the receptor gate at the channel pore. GABAAR gating is complex, comprising three major categories of transitions: openings/closings, preactivation, and desensitization. Interestingly, mutations at, e.g., the ligand binding site affect not only binding but often also more than one gating category, suggesting that structural determinants for distinct conformational transitions are shared. Gielen and co-workers (2015) proposed that the GABAAR desensitization gate is located at the second and third transmembrane segment. However, studies of our and others' groups indicated that other parts of the GABAAR macromolecule might be involved in this process. In the present study, we asked how selected point mutations (ß2G254V, α1G258V, α1L300V, and ß2L296V) at the M2 and M3 transmembrane segments affect gating transitions of the α1ß2γ2 GABAAR. Using high resolution macroscopic and single-channel recordings and analysis, we report that these substitutions, besides affecting desensitization, also profoundly altered openings/closings, having some minor effect on preactivation and agonist binding. Thus, the M2 and M3 segments primarily control late gating transitions of the receptor (desensitization, opening/closing), providing a further support for the concept of diffuse gating mechanisms for conformational transitions of GABAAR.

Induction of Inhibitory Synaptic Plasticity Enhances Tonic Current by Increasing the Content of α5-Subunit Containing GABAA Receptors in Hippocampal Pyramidal Neurons.

It is known that besides synaptic inhibition, there is a persistent component of inhibitory drive mediated by tonic currents which is believed to mediate majority of the total inhibitory charge in hippocampal neurons. Tonic currents, depending on cell types, can be mediated by a variety of GABAA receptor (GABAAR) subtypes but in pyramidal neurons, α5-subunit containing receptors were found to be predominant. Importantly, α5-GABAARs were implicated in both inhibitory and excitatory synaptic plasticity as well as in a variety of cognitive tasks. In the present study, we asked whether the protocol that evokes NMDAR-dependent GABAergic inhibitory long-term potentiation (iLTP) also induces the plasticity of tonic inhibition in hippocampal pyramidal neurons. Our whole-cell patch-clamp recordings revealed that the induction of this type of iLTP is associated with a marked increase in tonic current. By using the specific inverse agonist of α5-containing GABAARs (L-655,709) we provide evidence that this plastic change in tonic current is correlated with an increased proportion of this type of GABAARs. On the contrary, the iLTP induction did not affect the tonic current potentiated by THIP, indicating that the pool of δ subunit-containing GABAARs receptors remains unaffected. We conclude that the α5-GABAARs-dependent plasticity of tonic inhibition is a novel dimension of the neuroplasticity of the inhibitory drive in the hippocampal principal neurons. Overall, α5-containing GABAARs emerge as key players in a variety of plasticity mechanisms operating over a large span of time and spatial scales.

α1 Subunit Histidine 55 at the Interface between Extracellular and Transmembrane Domains Affects Preactivation and Desensitization of the GABAA Receptor.

The GABAA 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 ß1-ß2 loop of the α1 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.

Long-term plasticity of inhibitory synapses in the hippocampus and spatial learning depends on matrix metalloproteinase 3.

Learning and memory are known to depend on synaptic plasticity. Whereas the involvement of plastic changes at excitatory synapses is well established, plasticity mechanisms at inhibitory synapses only start to be discovered. Extracellular proteolysis is known to be a key factor in glutamatergic plasticity but nothing is known about its role at GABAergic synapses. We reveal that pharmacological inhibition of MMP3 activity or genetic knockout of the Mmp3 gene abolishes induction of postsynaptic iLTP. Moreover, the application of exogenous active MMP3 mimics major iLTP manifestations: increased mIPSCs amplitude, enlargement of synaptic gephyrin clusters, and a decrease in the diffusion coefficient of synaptic GABAA receptors that favors their entrapment within the synapse. Finally, we found that MMP3 deficient mice show faster spatial learning in Morris water maze and enhanced contextual fear conditioning. We conclude that MMP3 plays a key role in iLTP mechanisms and in the behaviors that presumably in part depend on GABAergic plasticity.

Interaction between GABAA receptor α1 and ß2 subunits at the N-terminal peripheral regions is crucial for receptor binding and gating.

Pentameric ligand gated ion channels (pLGICs) are crucial in electrochemical signaling but exact molecular mechanisms of their activation remain elusive. So far, major effort focused on the top-down molecular pathway between the ligand binding site and the channel gate. However, recent studies revealed that pLGIC activation is associated with coordinated subunit twisting in the membrane plane. This suggests a key role of intersubunit interactions but the underlying mechanisms remain largely unknown. Herein, we investigated a "peripheral" subunit interface region of GABAA receptor where structural modeling indicated interaction between N-terminal α1F14 and ß2F31 residues. Our experiments underscored a crucial role of this interaction in ligand binding and gating, especially preactivation and opening, showing that the intersubunit cross-talk taking place outside (above) the top-down pathway can be strongly involved in receptor activation. Thus, described here intersubunit interaction appears to operate across a particularly long distance, affecting vast portions of the macromolecule.

Mutations of α1F45 residue of GABAA receptor loop G reveal its involvement in agonist binding and channel opening/closing transitions.

GABAA receptors (GABAARs) mediate inhibitory neurotransmission in the mammalian brain. Recently, numerous GABAAR static structures have been published, but the molecular mechanisms of receptor activation remain elusive. Loop G is a rigid ß-strand belonging to an extensive ß-sheet that spans the regions involved in GABA binding and the interdomain interface which is important in receptor gating. It has been reported that loop G participates in ligand binding and gating of GABAARs, however, it remains unclear which specific gating transitions are controlled by this loop. Analysis of macroscopic responses revealed that mutation at the α1F45 residue (loop G midpoint) resulted in slower macroscopic desensitization and accelerated deactivation. Single-channel analysis revealed that these mutations also affected open and closed times distributions and reduced open probability. Kinetic modeling demonstrated that mutations affected primarily channel opening/closing and ligand binding with a minor effect on preactivation. Thus, α1F45 residue, in spite of its localization close to binding site, affects late gating transitions. In silico structural analysis suggested an important role of α1F45 residue in loop G stability and rigidity as well as in general structure of the binding site. We propose that the rigid ß-sheet comprising loop G is well suited for long range communication within GABAAR but this mechanism becomes impaired when α1F45 is mutated. In conclusion, we demonstrate that loop G is crucial in controlling both binding and gating of GABAARs. These data shed new light on GABAAR activation mechanism and may also be helpful in designing clinically relevant modulators.

The C loop at the orthosteric binding site is critically involved in GABAA receptor gating.

GABAA receptors (GABAARs) play a crucial role in mammalian adult brain inhibition. The dysfunction of GABAergic drive is related to such disorders as epilepsy, schizophrenia, and depression. Substantial progress has recently been made in describing the static structure of GABAARs, but the molecular mechanisms that underlie the activation process remain elusive. The C loop of the GABAAR structure shows the largest movement upon ligand binding to the orthosteric binding site, a phenomenon that is referred to as "capping." The C loop is known to be involved in agonist binding, but its role in the gating of Cys-loop receptors is still debated. Herein, we investigated this issue by analyzing the impact of a ß2F200 residue mutation of the C loop on gating properties of α1ß2γ2 GABAARs. Extensive analyses and the modeling of current responses to saturating agonist application demonstrated that this mutation strongly affected preactivation, opening, closing and desensitization, i.e. all considered gating steps. Single-channel analysis revealed that the ß2F200 mutation slowed all shut time components, and open times were shortened. Model fitting of these single-channel data further confirmed that the ß2F200 mutation strongly affected all of the gating characteristics. We also found that this mutation altered receptor sensitivity to the benzodiazepine flurazepam, which was attributable to a change in preactivation kinetics. In silico analysis indicated that the ß2F200 mutation resulted in distortion of the C loop structure, causing the movement of its tip from the binding site. Altogether, we provide the first evidence that C loop critically controls GABAAR gating.

Synaptic Potentiation at Basal and Apical Dendrites of Hippocampal Pyramidal Neurons Involves Activation of a Distinct Set of Extracellular and Intracellular Molecular Cues.

In the central nervous system, several forms of experience-dependent plasticity, learning and memory require the activity-dependent control of synaptic efficacy. Despite substantial progress in describing synaptic plasticity, mechanisms related to heterogeneity of synaptic functions at local circuits remain elusive. Here we studied the functional and molecular aspects of hippocampal circuit plasticity by analyzing excitatory synapses at basal and apical dendrites of mouse hippocampal pyramidal cells (CA1 region) in acute brain slices. In the past decade, activity of metalloproteinases (MMPs) has been implicated as a widespread and critical factor in plasticity mechanisms at various projections in the CNS. However, in the present study we discovered that in striking contrast to apical dendrites, synapses located within basal dendrites undergo MMP-independent synaptic potentiation. We demonstrate that synapse-specific molecular pathway allowing MMPs to rapidly upregulate function of NMDARs in stratum radiatum involved protease activated receptor 1 and intracellular kinases and GTPases activity. In contrast, MMP-independent scaling of synaptic strength in stratum oriens involved dopamine D1/D5 receptors and Src kinases. Results of this study reveal that 2 neighboring synaptic systems differ significantly in extracellular and intracellular cascades that control synaptic gain and provide long-searched transduction pathways relevant for MMP-dependent synaptic plasticity.

Protons modulate gating of recombinant α1ß2γ2 GABAA receptor by affecting desensitization and opening transitions.

Protons are potent modulators of GABAA receptors (GABAARs) and α1Phe64 residue was implicated in their pH sensitivity. Recently, we have demonstrated that this residue is involved in flipping transitions which precede channel opening. We thus re-addressed the mechanism of GABAAR modulation by protons by considering the gating scheme extended by flipping. The impact of pH changes was examined on currents mediated by wild-type α1ß2γ2 receptors or by their α1Phe64Leu or α1Phe64Cys mutants and elicited by saturating concentrations of full (GABA) or partial (piperidine-4-sulfonic acid) agonists. To describe the impact of extracellular pH on receptor gating, we combined macroscopic analysis of currents elicited by rapid agonist applications with single-channel studies. Acidification (pH 6.0) increased current amplitudes (in the case of leucine mutants effect was stronger when P4S was used) and decreased the rate and the extent of desensitization whereas alkalization (pH 8.0) had the opposite but weaker effect. Deactivation kinetics for wild-type receptors was slowed down by acidification while in the case of mutants this effect was observed upon alkalization. Moreover, α1Phe64 mutations enhanced GABAAR sensitivity to alkaline pH. Single-channel analysis revealed that acidification prolonged burst durations and affected shut but not open time distributions. Model simulations for macroscopic and single-channel activity indicated a novel mechanism in which protons primarily affected opening and desensitization rates but not flipping/unflipping. This evidence for the impact of protons on the receptor gating together with previously demonstrated effect on the agonist binding, point to a complex effect of extracellular pH on GABAAR macromolecule.

MMP­3 deficiency does not influence the length and number of CA1 dendrites of hippocampus of adult mice.

Over the past two decades, metalloproteinases (MMPs), including MMP­2, MMP­3, and MMP­9, have been implicated as important players in mechanisms underlying various forms of neuroplasticity. In particular, MMP­3 was found to be involved in both cognitive functions and in plasticity phenomena, but the underlying molecular mechanisms remain largely elusive. In general, it is believed that functional plasticity of neurons is associated with morphological alterations. Interestingly, MMP­9, in addition to playing a key role in synaptic plasticity, was found to affect plasticity­related spine morphology changes. Whereas the involvement of MMP­3 in shaping synapse morphology upon induction of synaptic plasticity awaits determination, it has been demostrated that MMP­3 knockout results in clearly altered apical dendrite morphology in pyramidal neurons in mouse visual cortex. Considering that the involvement of MMP­3 in synaptic plasticity has been most extensively documented for the CA1 hippocampal region, we decided to investigate whether genetic deletion of MMP­3 affects neuronal morphology in this area. To this end, we used Golgi staining to compare dendritic morphology of pyramidal neurons in the CA1 region in MMP­3­deficient and wild­type mice. Surprisingly, in contrast to the results obtained in cortex, extensive analysis of dendritic morphology in the CA1 region revealed no significant differences between MMP­3 knockout and wild­type groups. These results suggest that the impact of MMP­3 on neuronal morphology may be region­specific.

Distinct Modulation of Spontaneous and GABA-Evoked Gating by Flurazepam Shapes Cross-Talk Between Agonist-Free and Liganded GABAA Receptor Activity.

GABAA receptors (GABAARs) play a crucial inhibitory role in the CNS. Benzodiazepines (BDZs) are positive modulators of specific subtypes of GABAARs, but the underlying mechanism remains obscure. Early studies demonstrated the major impact of BDZs on binding and more recent investigations indicated gating, but it is unclear which transitions are affected. Moreover, the upregulation of GABAAR spontaneous activity by BDZs indicates their impact on receptor gating but the underlying mechanisms remain unknown. Herein, we investigated the effect of a BDZ (flurazepam) on the spontaneous and GABA-induced activity for wild-type (WT, α1ß2γ2) and mutated (at the orthosteric binding site α1F64) GABAARs. Surprisingly, in spite of the localization at the binding site, these mutations increased the spontaneous activity. Flurazepam (FLU) upregulated this activity for mutants and WT receptors to a similar extent by affecting opening/closing transitions. Spontaneous activity affected GABA-evoked currents and is manifested as an overshoot after agonist removal that depended on the modulation by BDZs. We explain the mechanism of this phenomenon as a cross-desensitization of ligand-activated and spontaneously active receptors. Moreover, due to spontaneous activity, FLU-pretreatment and co-application (agonist + FLU) protocols yielded distinct results. We provide also the first evidence that GABAAR may enter the desensitized state in the absence of GABA in a FLU-dependent manner. Based on our data and model simulations, we propose that FLU affects agonist-induced gating by modifying primarily preactivation and desensitization. We conclude that the mechanisms of modulation of spontaneous and ligand-activated GABAAR activity concerns gating but distinct transitions are affected in spontaneous and agonist-evoked activity.