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Significance Phosphodiesterase 10A (PDE10A) is as a target of interest in Huntington’s disease (HD) as levels of the enzyme have been shown to decrease prior to the development of the hallmark motor symptoms. Clearly, a better understanding of how PDE10A protein levels change as HD develops is required. Here we show that mutations in the regulatory GAF domains of PDE10A that cause hyperkinetic syndromes in humans lead to misprocessing of the PDE10A enzyme that ultimately leads to targeted degradation by the ubiquitin proteasome system or clearance by autophagy. Both mechanisms result in a paucity of PDE10A activity that lead to a loss of movement coordination. Our research suggests that similar mechanisms may underpin PDE10A loss during HD., A robust body of evidence supports the concept that phosphodiesterase 10A (PDE10A) activity in the basal ganglia orchestrates the control of coordinated movement in human subjects. Although human mutations in the PDE10A gene manifest in hyperkinetic movement disorders that phenocopy many features of early Huntington’s disease, characterization of the maladapted molecular mechanisms and aberrant signaling processes that underpin these conditions remains scarce. Recessive mutations in the GAF-A domain have been shown to impair PDE10A function due to the loss of striatal PDE10A protein levels, but here we show that this paucity is caused by irregular intracellular trafficking and increased PDE10A degradation in the cytosolic compartment. In contrast to GAF-A mutants, dominant mutations in the GAF-B domain of PDE10A induce PDE10A misfolding, a common pathological phenotype in many neurodegenerative diseases. These data demonstrate that the function of striatal PDE10A is compromised in disorders where disease-associated mutations trigger a reduction in the fidelity of PDE compartmentalization.

First-in-line benzodiazepine treatment fails to terminate seizures in about 30% of epilepsy patients, highlighting a need for novel antiseizure strategies. Impaired GABAergic inhibition is key to the development of such benzodiazepine-resistant seizures, as well as the pathophysiology of status epilepticus (SE). It is emerging that reduced or impaired neuronal K+/Cl- cotransporter 2 (KCC2) activity contributes to deficits in γ-aminobutyric acid (GABA)-mediated inhibition and increased seizure vulnerability. The with-no-lysine kinase (WNK)-STE20/SPS1-related proline/alanine-rich (SPAK) kinase signaling pathway inhibits neuronal KCC2 via KCC2-T1007 phosphorylation. A selective WNK kinase inhibitor, WNK463, was recently synthesized by Novartis. Exploiting WNK463, we test the hypothesis that pharmacological WNK inhibition will enhance KCC2 activity, increase the efficacy of GABAergic inhibition, and thereby limit seizure activity in animal models. Immunoprecipitation and Western blot analysis were used to examine WNK463's effects on KCC2-T1007 phosphorylation, in vitro and in vivo. A thallium (Tl+) uptake assay was used in human embryonic kidney (HEK-293) cells expressing KCC2 to test WNK463's effects on KCC2-mediated Tl+ transport. Gramicidin-perforated- and whole-cell patch-clamp recordings in cortical rat neurons were used to examine WNK463's effects on KCC2-mediated Cl- transport. In mouse brain slices (entorhinal cortex), field recordings were utilized to examine WNK463's effects on 4-aminopyridine-induced seizure activity. Last, WNK463 was directly deliver to the mouse hippocampus in vivo and tested in a kainic acid model of diazepam-resistant SE. WNK463 significantly reduces KCC2-T1007 phosphorylation in vitro and in vivo (mice). In human embryonic kidney 293 (HEK-293) cells expressing KCC2, WNK463 greatly enhanced the rates Tl+ transport. However, the drug did not enhance Tl+ transport in cells expressing a KCC2-phospho null T1007 mutant. In cultured rat neurons, WNK463 rapidly reduced intracellular Cl- and consequently hyperpolarized the Cl- reversal potential (EGABA). In mature neurons that were artificially loaded with 30 mM Cl-, WNK463 significantly enhanced KCC2-mediated Cl- export and hyperpolarized EGABA. In a 4-aminopyridine model of acute seizures, WNK463 reduced the frequency and number of seizure-like events (SLEs). Finally, in an in vivo kainic acid (KA) model of diazepam-resistant SE, WNK463 slowed the onset and reduced the severity of KA-induced status epilepticus. Last, WNK463 prevented the development of pharmaco-resistance to diazepam in drug-treated mice. Our findings demonstrate that acute WNK463 treatment potentiates KCC2 activity in neurons and limits seizure burden in two well-established models of seizures and epilepsy. Our work suggests that agents which act to increase KCC2 activity may be useful adjunct therapeutics to alleviate diazepam-resistant SE.

KCC2 plays a critical role in determining the efficacy of synaptic inhibition and deficits in its activity lead to epilepsy and neurodevelopmental delay. Here we use unbiased proteomic analyses to demonstrate that KCC2 forms stable protein complexes in the neuronal plasma membrane with 96 autism and/or epilepsy risk gene (ASD/Epi) products including ANKB, ANKG, CNTN1, ITPR1, NCKAP1, SCN2A, SHANK3, SPTAN1, and SPTBN1. Many of these proteins are also targets of Fragile-X mental retardation protein (FMRP), the inactivation of which is the leading monogenic cause of autism. Accordingly, the expression of a subset of these KCC2-binding partners was decreased in Fmr1 knockout mice. Fmr1 knockout compromised KCC2 phosphorylation, a key regulatory mechanism for transporter activity and the postnatal development of GABAergic inhibition. Thus, KCC2 is a point of convergence for multiple ASD/Epi risk genes and therapies targeting this transporter may have broad utility in alleviating these heterogeneous disorders and their associated epilepsies.

Mesial temporal lobe epilepsy (mTLE) is the most common form of epilepsy, believed to arise in part from compromised GABAergic inhibition. The neuronal specific K+/Cl− co-transporter 2 (KCC2) is a critical determinant of the efficacy of GABAergic inhibition and deficits in its activity are observed in mTLE patients and animal models of epilepsy. To test if reductions of KCC2 activity directly contribute to the pathophysiology of mTLE, we locally ablated KCC2 expression in a subset of principal neurons within the adult hippocampus. Deletion of KCC2 resulted in compromised GABAergic inhibition and the development of spontaneous, recurrent generalized seizures. Moreover, local ablation of KCC2 activity resulted in hippocampal sclerosis, a key pathological change seen in mTLE. Collectively, our results demonstrate that local deficits in KCC2 activity within the hippocampus are sufficient to precipitate mTLE. Keywords: KCC2, GABA, Epilepsy, Hippocampal sclerosis

apoE is the primary lipid carrier within the CNS and the strongest genetic risk factor for late onset Alzheimer's disease (AD). apoE is primarily lipidated via ABCA1, and both are under transcriptional regulation by the nuclear liver X receptor (LXR). Considerable evidence from genetic (using ABCA1 overexpression) and pharmacological (using synthetic LXR agonists) studies in AD mouse models suggests that increased levels of lipidated apoE can improve cognitive performance and, in some strains, can reduce amyloid burden. However, direct synthetic LXR ligands have hepatotoxic side effects that limit their clinical use. Here, we describe a set of small molecules, previously annotated as antagonists of the purinergic receptor, P2X7, which enhance ABCA1 expression and activity as well as apoE secretion, and are not direct LXR ligands. Furthermore, P2X7 is not required for these molecules to induce ABCA1 upregulation and apoE secretion, demonstrating that the ABCA1 and apoE effects are mechanistically independent of P2X7 inhibition. Hence, we have identified novel dual activity compounds that upregulate ABCA1 across multiple CNS cell types, including human astrocytes, pericytes, and microglia, through an indirect LXR mechanism and that also independently inhibit P2X7 receptor activity.

K+/Cl− cotransporter 2 (KCC2) is selectively expressed in the adult nervous system and allows neurons to maintain low intracellular Cl− levels. Thus, KCC2 activity is an essential prerequisite for fast hyperpolarizing synaptic inhibition mediated by type A γ-aminobutyric acid (GABAA) receptors, which are Cl−-permeable, ligand-gated ion channels. Consistent with this, deficits in the activity of KCC2 lead to epilepsy and are also implicated in neurodevelopmental disorders, neuropathic pain, and schizophrenia. Accordingly, there is significant interest in developing activators of KCC2 as therapeutic agents. To provide insights into the cellular processes that determine KCC2 activity, we have investigated the mechanism by which N-ethylmaleimide (NEM) enhances transporter activity using a combination of biochemical and electrophysiological approaches. Our results revealed that, within 15 min, NEM increased cell surface levels of KCC2 and modulated the phosphorylation of key regulatory residues within the large cytoplasmic domain of KCC2 in neurons. More specifically, NEM increased the phosphorylation of serine 940 (Ser-940), whereas it decreased phosphorylation of threonine 1007 (Thr-1007). NEM also reduced with no lysine (WNK) kinase phosphorylation of Ste20-related proline/alanine-rich kinase (SPAK), a kinase that directly phosphorylates KCC2 at residue Thr-1007. Mutational analysis revealed that Thr-1007 dephosphorylation mediated the effects of NEM on KCC2 activity. Collectively, our results suggest that compounds that either increase the surface stability of KCC2 or reduce Thr-1007 phosphorylation may be of use as enhancers of KCC2 activity.

KCC2 is a neuron specific K+-Cl− co-transporter that controls neuronal chloride homeostasis, and is critically involved in many neurological diseases including brain trauma, epilepsies, autism and schizophrenia. Despite significant accumulating data on the biology and electrophysiological properties of KCC2, structure-function relationships remain poorly understood. Here we used calixarene detergent to solubilize and purify wild-type non-aggregated and homogenous KCC2. Specific binding of inhibitor compound VU0463271 was demonstrated using surface plasmon resonance (SPR). Mass spectrometry revealed glycosylations and phosphorylations as expected from functional KCC2. We show by electron microscopy (EM) that KCC2 exists as monomers and dimers in solution. Monomers are organized into “head” and “core” domains connected by a flexible “linker”. Dimers are asymmetrical and display a bent “S-shape” architecture made of four distinct domains and a flexible dimerization interface. Chemical crosslinking in reducing conditions shows that disulfide bridges are involved in KCC2 dimerization. Moreover, we show that adding a tag to the C-terminus is detrimental to KCC2 function. We postulate that the conserved KCC2 C-ter may be at the interface of dimerization. Taken together, our findings highlight the flexible multi-domain structure of KCC2 with variable anchoring points at the dimerization interface and an important C-ter extremity providing the first in-depth functional architecture of KCC2.

Estrogen plays a critical role in many physiological processes and exerts profound effects on behavior by regulating neuronal excitability. While estrogen has been established to exert effects on dendritic morphology and excitatory neurotransmission its role in regulating neuronal inhibition is poorly understood. Fast synaptic inhibition in the adult brain is mediated by specialized populations of γ-c aA receptors (GABAARs) that are selectively enriched at synapses, a process dependent upon their interaction with the inhibitory scaffold protein gephyrin. Here we have assessed the role that estradiol (E2) plays in regulating the dynamics of GABAARs and stability of inhibitory synapses. Treatment of cultured cortical neurons with E2 reduced the accumulation of GABAARs and gephyrin at inhibitory synapses. However, E2 exposure did not modify the expression of either the total or the plasma membrane GABAARs or gephyrin. Mechanistically, single-particle tracking revealed that E2 treatment selectively reduced the dwell time and thereby decreased the confinement of GABAARs at inhibitory synapses. Consistent with our cell biology measurements, we observed a significant reduction in amplitude of inhibitory synaptic currents in both cultured neurons and hippocampal slices exposed to E2, while their frequency was unaffected. Collectively, our results suggest that acute exposure of neurons to E2 leads to destabilization of GABAARs and gephyrin at inhibitory synapses, leading to reductions in the efficacy of GABAergic inhibition via a postsynaptic mechanism.

The combination of in vitro multi-electrode arrays (MEAs) and the neuronal differentiation of stem cells offers the capability to study human neuronal networks from patient or engineered human cell lines. Here, we use MEA-based assays to probe synaptic function and network interactions of hiPSC-derived neurons. Neuronal network behaviour first emerges at approximately 30 days of culture and is driven by glutamate neurotransmission. Over a further 30 days, inhibitory GABAergic signalling shapes network behaviour into a synchronous regular pattern of burst firing activity and low activity periods. Gene mutations in L-type voltage gated calcium channel subunit genes are strongly implicated as genetic risk factors for the development of schizophrenia and bipolar disorder. We find that, although basal neuronal firing rate is unaffected, there is a dose-dependent effect of L-type voltage gated calcium channel inhibitors on synchronous firing patterns of our hiPSC-derived neural networks. This demonstrates that MEA assays have sufficient sensitivity to detect changes in patterns of neuronal interaction that may arise from hypo-function of psychiatric risk genes. Our study highlights the utility of in vitro MEA based platforms for the study of hiPSC neural network activity and their potential use in novel compound screening.

Impaired neuronal inhibition has long been associated with the increased probability of seizure occurrence and heightened seizure severity. Fast synaptic inhibition in the brain is primarily mediated by the type A γ-aminobutyric acid receptors (GABAARs), ligand-gated ion channels that can mediate Cl(-) influx resulting in membrane hyperpolarization and the restriction of neuronal firing. In most adult brain neurons, the K(+)/Cl(-) co-transporter-2 (KCC2) establishes hyperpolarizing GABAergic inhibition by maintaining low [Cl(-)]i. In this study, we sought to understand how decreased KCC2 transport function affects seizure event severity. We impaired KCC2 transport in the 0-Mg(2+) ACSF and 4-aminopyridine in vitro models of epileptiform activity in acute mouse brain slices. Experiments with the selective KCC2 inhibitor VU0463271 demonstrated that reduced KCC2 transport increased the duration of SLEs, resulting in non-terminating discharges of clonic-like activity. We also investigated slices obtained from the KCC2-Ser940Ala (S940A) point-mutant mouse, which has a mutation at a known functional phosphorylation site causing behavioral and cellular deficits under hyperexcitable conditions. We recorded from the entorhinal cortex of S940A mouse brain slices in both 0-Mg(2+) ACSF and 4-aminopyridine, and demonstrated that loss of the S940 residue increased the susceptibility of continuous clonic-like discharges, an in vitro form of status epilepticus. Our experiments revealed KCC2 transport activity is a critical factor in seizure event duration and mechanisms of termination. Our results highlight the need for therapeutic strategies that potentiate KCC2 transport function in order to decrease seizure event severity and prevent the development of status epilepticus.

The zinc-activated channel (ZAC) is a cationic ion channel belonging to the superfamily of Cys-loop receptors, which consists of pentameric ligand-gated ion channels. ZAC is the least understood member of this family so in the present study we sought to characterize the properties of this channel further. We demonstrate that not only zinc (Zn(2+)) but also copper (Cu(2+)) and protons (H(+)) are agonists of ZAC, displaying potencies and efficacies in the rank orders of H(+)>Cu(2+)>Zn(2+) and H(+)>Zn(2+)>Cu(2+), respectively. The responses elicited by Zn(2+), Cu(2+) and H(+) through ZAC are all characterized by low degrees of desensitization. In contrast, currents evoked by high concentrations of the three agonists comprise distinctly different activation and decay components, with transitions to and from an open state being significantly faster for H(+) than for the two metal ions. The permeabilities of ZAC for Na(+) and K(+) relative to Cs(+) are indistinguishable, whereas replacing all of extracellular Na(+) and K(+) with the divalent cations Ca(2+) or Mg(2+) results in complete elimination of Zn(2+)-activated currents at both negative and positive holding potentials. This indicates that ZAC is non-selectively permeable to monovalent cations, whereas Ca(2+) and Mg(2+) inhibit the channel. In conclusion, this is the first report of a Cys-loop receptor being gated by Zn(2+), Cu(2+) and H(+). ZAC could be an important mediator of some of the wide range of physiological functions regulated by or involving Zn(2+), Cu(2+) and H(+).

The type 2 K(+)/Cl(−) cotransporter (KCC2) allows neurons to maintain low intracellular levels of Cl(−), a prerequisite for efficient synaptic inhibition. Reductions in KCC2 activity are evident in epilepsy; however, whether these deficits directly contribute to the underlying pathophysiology remains controversial. To address this issue, we created knock-in mice in which threonines 906 and 1007 within KCC2 have been mutated to alanines (KCC2-T906A/T1007A), which prevents its phospho-dependent inactivation. The respective mice appeared normal and did not show any overt phenotypes, and basal neuronal excitability was unaffected. KCC2-T906A/T1007A mice exhibited increased basal neuronal Cl(−) extrusion, without altering total or plasma membrane accumulation of KCC2. Critically, activity-induced deficits in synaptic inhibition were reduced in the mutant mice. Consistent with this, enhanced KCC2 was sufficient to limit chemoconvulsant-induced epileptiform activity. Furthermore, this increase in KCC2 function mitigated induction of aberrant high-frequency activity during seizures, highlighting depolarizing GABA as a key contributor to the pathological neuronal synchronization seen in epilepsy. Thus, our results demonstrate that potentiating KCC2 represents a therapeutic strategy to alleviate seizures.

Fast inhibitory synaptic transmission is mediated by γ-aminobutyric acid type A receptors (GABAARs) that are enriched at functionally diverse synapses via mechanisms that remain unclear. Using isothermal titration calorimetry and complementary methods we demonstrate an exclusive low micromolar binding of collybistin to the α2-subunit of GABAARs. To explore the biological relevance of collybistin-α2-subunit selectivity, we generate mice with a mutation in the α2-subunit-collybistin binding region (Gabra2-1). The mutation results in loss of a distinct subset of inhibitory synapses and decreased amplitude of inhibitory synaptic currents. Gabra2–1 mice have a striking phenotype characterized by increased susceptibility to seizures and early mortality. Surviving Gabra2-1 mice show anxiety and elevations in electroencephalogram δ power, which are ameliorated by treatment with the α2/α3-selective positive modulator, AZD7325. Taken together, our results demonstrate an α2-subunit selective binding of collybistin, which plays a key role in patterned brain activity, particularly during development.

Our knowledge of how estrogen signaling can influence inhibitory synaptic transmission is rudimentary and is addressed here. Collectively, our data suggest that estrogen modulates the dynamics of surface GABAA receptors and hence efficacy of synaptic inhibition, via a postsynaptic mechanism that relies on disrupting the postsynaptic scaffold. This regulatory mechanism may have profound effects on the efficacy of neuronal inhibition and therefore synaptic plasticity and thus play a role in the pathophysiology of estrogen-related seizure and cognitive disorders.

Deficits in GABAergic inhibition result in the abnormal neuronal activation and synchronization that underlies seizures. However, the molecular mechanisms responsible for transforming a normal brain into an epileptic one remain largely unknown. Hyperpolarizing inhibition mediated by type A GABA (GABAA) receptors is dependent on chloride extrusion by the neuron-specific type 2K+-Cl- cotransporter (KCC2). Loss-of-function mutations in KCC2 are a known cause of infantile epilepsy in humans and KCC2 dysfunction is present in patients with both idiopathic and acquired epilepsy. Here we discuss the growing evidence that KCC2 dysfunction has a central role in the development and severity of the epilepsies.

Mutations in the gene TARDBP, which encodes TAR DNA-binding protein 43 (TDP-43), are a rare cause of familial forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). While the majority of mutations are found in the C-terminal glycine-rich domain, an alanine to valine amino acid change at position 90 (A90V) in the bipartite nuclear localization signal of TDP-43 has been described. This sequence variant has previously been shown to cause cytoplasmic mislocalization of TDP-43 and decreases protein solubility, leading to the formation of insoluble aggregates. Since the A90V mutation has been described both in patients as well as healthy controls, its pathogenic potential in ALS and FTD remains unclear. Here we compare properties of overexpressed A90V to the highly pathogenic M337V mutation. Though both mutations drive mislocalization of the protein to the cytoplasm to the same extent, M337V produces more significant damage in terms of protein solubility, levels of pathogenic phosphorylation, and formation of C-terminal truncated protein species. Furthermore, the M337V, but not the A90V mutant, leads to a downregulation of histone deacetylase 6 (HDAC6) and Ras GTPase-activating protein-binding protein (G3BP). We hypothesize that the A90V variant represents a genetic risk factor for ALS and FTD, that by itself is not sufficient to cause the full repertoire of deleterious phenotypes associated with TDP-43 proteinopathies, despite prominent cytoplasmic protein relocalization.

The KCC2 cotransporter establishes the low neuronal Cl(-) levels required for GABAA and glycine (Gly) receptor-mediated inhibition, and KCC2 deficiency in model organisms results in network hyperexcitability. However, no mutations in KCC2 have been documented in human disease. Here, we report two non-synonymous functional variants in human KCC2, R952H and R1049C, exhibiting clear statistical association with idiopathic generalized epilepsy (IGE). These variants reside in conserved residues in the KCC2 cytoplasmic C-terminus, exhibit significantly impaired Cl(-)-extrusion capacities resulting in less hyperpolarized Gly equilibrium potentials (EG ly), and impair KCC2 stimulatory phosphorylation at serine 940, a key regulatory site. These data describe a novel KCC2 variant significantly associated with a human disease and suggest genetically encoded impairment of KCC2 functional regulation may be a risk factor for the development of human IGE.

The 5-HT3A receptor homology model, based on the partial structure of the nicotinic acetylcholine receptor from Torpedo marmorata, reveals an asymmetric ion channel with five portals framed by adjacent helical amphipathic (HA) stretches within the 114-residue loop between the M3 and M4 membrane-spanning domains. The positive charge of Arg-436, located within the HA stretch, is a rate-limiting determinant of single channel conductance (γ). Further analysis reveals that positive charge and volume of residue 436 are determinants of 5-HT3A receptor inward rectification, exposing an additional role for portals. A structurally unresolved stretch of 85 residues constitutes the bulk of the M3-M4 loop, leaving a >45-A gap in the model between M3 and the HA stretch. There are no additional structural data for this loop, which is vestigial in bacterial pentameric ligand-gated ion channels and was largely removed for crystallization of the Caenorhabditis elegans glutamate-activated pentameric ligand-gated ion channels. We created 5-HT3A subunit loop truncation mutants, in which sequences framing the putative portals were retained, to determine the minimum number of residues required to maintain their functional integrity. Truncation to between 90 and 75 amino acids produced 5-HT3A receptors with unaltered rectification. Truncation to 70 residues abolished rectification and increased γ. These findings reveal a critical M3-M4 loop length required for functions attributable to cytoplasmic portals. Examination of all 44 subunits of the human neurotransmitter-activated Cys-loop receptors reveals that, despite considerable variability in their sequences and lengths, all M3-M4 loops exceed 70 residues, suggesting a fundamental requirement for portal integrity.

Benzodiazepines have been used for decades as first-line treatment for status epilepticus (SE). For reasons that are not fully understood, the efficacy of benzodiazepines decreases with increasing duration of seizure activity. This often forces clinicians to resort to more drastic second- and third-line treatments that are not always successful. The antiseizure properties of benzodiazepines are mediated by γ-aminobutyric acid type A (GABA(A) ) receptors. Decades of research have focused on the failure of GABAergic inhibition after seizure onset as the likely cause of the development benzodiazepine resistance during SE. However, the details of the deficits in GABA(A) signaling are still largely unknown. Therefore, it is necessary to improve our understanding of the mechanisms of benzodiazepine resistance so that more effective strategies can be formulated. In this review we discuss evidence supporting the role of altered GABA(A) receptor function as the major underlying cause of benzodiazepine-resistant SE in both humans and animal models. We specifically address the prevailing hypothesis, which is based on changes in the number and subtypes of GABA(A) receptors, as well as the potential influence of perturbed chloride homeostasis in the mature brain.

The accumulation of γ-aminobutyric acid receptors (GABAARs) at the appropriate postsynaptic sites is critical for determining the efficacy of fast inhibitory neurotransmission. Although we know that the majority of synaptic GABAAR subtypes are assembled from α1–3, β, and γ2 subunits, our understanding of how neurons facilitate their targeting to and stabilization at inhibitory synapses is rudimentary. To address these issues, we have created knock-in mice in which the pH-sensitive green fluorescent protein (GFP) and the Myc epitope were introduced to the extracellular domain of the mature receptor α2 subunit (pHα2). Using immunoaffinity purification and mass spectroscopy, we identified a stable complex of 174 proteins that were associated with pHα2, including other GABAAR subunits, and previously identified receptor-associated proteins such as gephyrin and collybistin. 149 of these proteins were novel GABAAR binding partners and included G-protein-coupled receptors and ion channel subunits, proteins that regulate trafficking and degradation, regulators of protein phosphorylation, GTPases, and a number of proteins that regulate their activity. Notably, members of the postsynaptic density family of proteins that are critical components of excitatory synapses were not associated with GABAARs. Crucially, we demonstrated for a subset of these novel proteins (including cullin1, ephexin, potassium channel tetramerization domain containing protein 12, mitofusin2, metabotropic glutamate receptor 5, p21-activated kinase 7, and Ras-related protein 5A) bind directly to the intracellular domains of GABAARs, validating our proteomic analysis. Thus, our experiments illustrate the complexity of the GABAAR proteome and enhance our understanding of the mechanisms neurons use to construct inhibitory synapses.

BACKGROUND AND PURPOSE Morphine is an antagonist at 5-HT3A receptors. 5-HT3 and opioid receptors are expressed in many of the same neuronal pathways where they modulate gut motility, pain and reinforcement. There is increasing interest in the 5-HT3B subunit, which confers altered pharmacology to 5-HT3 receptors. We investigated the mechanisms of inhibition by morphine of 5-HT3 receptors and the influence of the 5-HT3B subunit. EXPERIMENTAL APPROACH 5-HT-evoked currents were recorded from voltage-clamped HEK293 cells expressing human 5-HT3A subunits alone or in combination with 5-HT3B subunits. The affinity of morphine for the orthosteric site of 5-HT3A or 5-HT3AB receptors was assessed using radioligand binding with the antagonist [3H]GR65630. KEY RESULTS When pre-applied, morphine potently inhibited 5-HT-evoked currents mediated by 5-HT3A receptors. The 5-HT3B subunit reduced the potency of morphine fourfold and increased the rates of inhibition and recovery. Inhibition by pre-applied morphine was insurmountable by 5-HT, was voltage-independent and occurred through a site outside the second membrane-spanning domain. When applied simultaneously with 5-HT, morphine caused a lower potency, surmountable inhibition of 5-HT3A and 5-HT3AB receptors. Morphine also fully displaced [3H]GR65630 from 5-HT3A and 5-HT3AB receptors with similar potency. CONCLUSIONS AND IMPLICATIONS These findings suggest that morphine has two sites of action, a low-affinity, competitive site and a high-affinity, non-competitive site that is not available when the channel is activated. The affinity of morphine for the latter is reduced by the 5-HT3B subunit. Our results reveal that morphine causes a high-affinity, insurmountable and subunit-dependent inhibition of human 5-HT3 receptors.

Picrotoxin and t-butylbicyclophosphorothionate (TBPS) are GABA(A) receptor (GABA(A)R) open channel blockers. However, picrotoxin displaceable [(35)S]TBPS binding to α1β2γ2 GABA(A)Rs occurs in the absence of GABA, suggesting that access to the binding site is independent of activation. Alternatively, spontaneous gating may provide access to the channel. In the absence of episodic GABA application, picrotoxin and TBPS blocked (by 91 ± 3% and 85 ± 5%, respectively) GABA-evoked currents mediated by α1β2γ2 receptors. We used two approaches to inhibit spontaneous GABA(A)R gating, bicuculline, which inhibits spontaneous current in the absence of exogenous agonist and the α1(K278M) mutant subunit. Whole-cell patch-clamp recordings demonstrated that α1(K278M)β2γ2 receptors have negligible spontaneous gating. Application of bicuculline to α1β2γ2 receptors in the absence of exogenous GABA caused a 35% reduction of current blockade by TBPS and reduced [(35)S]TBPS binding by 25%. Consistent with this, in the absence of exogenous GABA, α1(K278M)β2γ2 receptors exhibited reduced blockade by TBPS current compared to wild-type receptors. These data suggest that a decrease in spontaneous gating reduces accessibility of TBPS to its binding site. GABA application during picrotoxin or TBPS administration enhanced α1β2γ2 receptor blockade (to 98% in both cases). The GABA-dependent component of TBPS blockade accounts for the stimulation of [(35)S]TBPS binding to α1β2γ2 receptors seen with GABA (1 μm) application. Moreover, application of GABA at concentrations that cause significant steady-state desensitization reduced [(35)S]TBPS binding. The α1(K278M) subunit slowed desensitization kinetics and increased the rate of deactivation of GABA-evoked currents. Furthermore, there was a marked increase in the GABA EC(50) for desensitization of α1(K278M)β2γ2 receptors associated with a large increase in the GABA-dependent stimulation of [(35)S]TBPS binding. These data establish a relationship between GABA(A)R function and the three phases of [(35)S]TBPS binding seen in the absence and the presence of GABA.

KCC2 comprises the major Cl(-) extruding mechanism in most adult neurons. Hyperpolarizing GABAergic transmission depends on KCC2 function. We recently demonstrated that glutamate reduces KCC2 function by a phosphorylation-dependent mechanism that leads to excitatory GABA responses. Here we investigated the methods by which to estimate changes in E(GABA), as well as the processes that lead to depolarizing GABA responses and their effects on neuronal excitability. We demonstrated that current-clamp recordings of membrane potential responses to GABA can determine upper and lower limits of E(GABA). We also further characterized depolarizing GABA responses, which both excited and inhibited neurons. Our analyses revealed that persistently active GABA(A) receptors contributed to loading Cl(-) during the glutamate exposure, indicating that tonic inhibition can facilitate the development of depolarizing GABA responses and increase excitability after pathophysiological insults. Finally, we demonstrated that hyperpolarizing GABA responses could temporarily switch to depolarizing responses when they coincided with an afterhyperpolarization.

SummaryCyclin E is a component of the core cell cycle machinery, and it drives cell proliferation by regulating entry and progression of cells through the DNA synthesis phase. Cyclin E expression is normally restricted to proliferating cells. However, high levels of cyclin E are expressed in the adult brain. The function of cyclin E in quiescent, postmitotic nervous system remains unknown. Here we use a combination of in vivo quantitative proteomics and analyses of cyclin E knockout mice to demonstrate that in terminally differentiated neurons cyclin E forms complexes with Cdk5 and controls synapse function by restraining Cdk5 activity. Ablation of cyclin E led to a decreased number of synapses, reduced number and volume of dendritic spines, and resulted in impaired synaptic plasticity and memory formation in cyclin E-deficient animals. These results reveal a cell cycle-independent role for a core cell cycle protein, cyclin E, in synapse function and memory.

The cysteine (Cys) residue at position 312 in the third transmembrane domain (M3) is conserved among 5-hydroxytryptamine type 3 (5-HT3) receptor subunits and many other subunits of the nicotinic acetylcholine (nACh) related Cys-loop receptor family, including most of the γ-aminobutyric acid type A (GABAA) and glycine receptor subunits. To elucidate a possible role for the Cys-312 in human 5-HT3A receptors, we replaced it with alanine and expressed the 5-HT3A(C312A) mutant in HEK293 cells. The mutation resulted in an absence of 5-HT-induced whole-cell current without reducing homopentamer formation, surface expression or 5-HT binding. The 5-HT3A(C312A) mutant, when co-expressed with the wild-type 5-HT3A subunit, did not affect functional expression of receptors, suggesting that the mutant is not dominant negative. Interestingly, co-expression of 5-HT3A(C312A) with 5-HT3B led to surface expression of heteropentamers that mediated small 5-HT responses. This suggests that the Cys-312 is essential for homomeric but not heteromeric receptor gating. To further investigate the relationship between residue 312 and gating we replaced it with amino acids located at the equivalent position within other Cys-loop subunits that are either capable or incapable of forming functional homopentamers. Replacement of 5-HT3A Cys-312 by Gly or Leu (equivalent residues in the nACh receptor δ and γ subunits) abolished and severely attenuated function, respectively, whereas replacement by Thr or Ser (equivalent residues in nACh receptor α7 and GABAAρ subunits) supported robust function. Thus, 5-HT3A residue 312 and equivalent polar residues in the M3 of other Cys-loop subunits are essential determinants of homopentameric gating.

Homomeric 5-hydroxytryptamine (5-HT)(3A) and heteromeric 5-HT(3AB) receptors mediate rapid excitatory responses to serotonin in the central and peripheral nervous systems. The alkaloid morphine, in addition to being a mu-opioid receptor agonist, is a potent competitive inhibitor of 5-HT(3) receptors. We examined whether methadone, an opioid often used to treat morphine dependence, also exhibited 5-HT(3) receptor antagonist properties. Racemic (R/S)-methadone inhibited currents mediated by human homomeric 5-HT(3A) receptors (IC(50) = 14.1 +/- 2.5 microM). Incorporation of the 5-HT(3B) subunit into heteromeric 5-HT(3AB) receptors reduced the potency of inhibition by (R/S)-methadone (IC(50) = 41.1 +/- 0.9 microM). (R/S)-Methadone also increased apparent desensitization of both 5-HT(3) receptor subtypes. The inhibition of the 5-HT(3A) receptor was competitive; however, incorporation of the 5-HT(3B) subunit caused the appearance of inhibition that was insurmountable by 5-HT. In the absence of rapid desensitization, when dopamine was used as an agonist of 5-HT(3AB) receptors, the inhibition by (R/S)-methadone was voltage-dependent. The antagonist and desensitization-enhancing effects of (R/S)-methadone were shared by pure (R)- and (S)-methadone enantiomers, which had similar actions on 5-HT-evoked currents mediated by 5-HT(3) receptors. However, (R)-methadone exhibited a larger voltage-dependent inhibition of dopamine-evoked currents mediated by 5-HT(3AB) receptors than did (S)-methadone. Inhibition of 5-HT(3A) receptors by (R/S)-methadone was not influenced by voltage. Thus, methadone displays multimodal subunit-dependent antagonism of 5-HT(3) receptors.

Cation-selective cysteine (Cys)-loop transmitter-gated ion channels provide an important pathway for Ca2+ entry into neurones. We examined the influence on Ca2+ permeation of amino acids located at intra- and extracellular ends of the conduction pathway of the human 5-hydroxytryptamine type 3A (5-HT3A) receptor. Mutation of cytoplasmic arginine residues 432, 436, and 440 to glutamine, aspartate, and alanine (the aligned residues of the human 5-HT3B subunit (yielding 5-HT3A(QDA)) increased PCa/PCs from 1.4 to 3.7. The effect was attributable to the removal of an electrostatic influence of the Arg-436 residue. Despite its relatively high permeability to Ca2+, the single channel conductance of the 5-HT3A(QDA) receptor was depressed in a concentration-dependent and voltage-independent manner by extracellular Ca2+. A conserved aspartate, located toward the extracellular end of the conduction pathway and known to influence ionic selectivity, contributed to the inhibitory effect of Ca2+ on macroscopic currents mediated by 5-HT3A receptors. We introduced a D293A mutation into the 5-HT3A(QDA) receptor (yielding the 5-HT3A(QDA D293A) construct) to determine whether the aspartate is required for the suppression of single channel conductance by Ca2+. The D293A mutation decreased the PCa/PCs ratio to 0.25 and reduced inwardly directed single channel conductance from 41 to 30 pS but did not prevent suppression of single channel conductance by Ca2+. The D293A mutation also reduced PCa/PCs when engineered into the wild-type 5-HT3A receptor. The data helped to identify key residues in the cytoplasmic domain (Arg-436) and extracellular vestibule (Asp-293) that markedly influence PCa/PCs and additionally directly demonstrated a depression of single channel conductance by Ca2+.

GABAAreceptors form Cl−permeable channels that mediate the majority of fast synaptic inhibition in the brain. The K+/Cl−cotransporter KCC2 is the main mechanism by which neurons establish low intracellular Cl−levels, which is thought to enable GABAergic inhibitory control of neuronal activity. However, the widely used KCC2 inhibitor furosemide is nonselective with antiseizure efficacy in slices andin vivo, leading to a conflicting scheme of how KCC2 influences GABAergic control of neuronal synchronization. Here we used the selective KCC2 inhibitor VU0463271 [N-cyclopropyl-N-(4-methyl-2-thiazolyl)-2-[(6-phenyl-3-pyridazinyl)thio]acetamide] to investigate the influence of KCC2 function. Application of VU0463271 caused a reversible depolarizing shift inEGABAvalues and increased spiking of cultured hippocampal neurons. Application of VU0463271 to mouse hippocampal slices under low-Mg2+conditions induced unremitting recurrent epileptiform discharges. Finally, microinfusion of VU0463271 alone directly into the mouse dorsal hippocampus rapidly caused epileptiform discharges. Our findings indicated that KCC2 function was a critical inhibitory factorex vivoandin vivo.

The K(+)/Cl(-) cotransporter (KCC2) allows adult neurons to maintain low intracellular Cl(-) levels, which are a prerequisite for efficient synaptic inhibition upon activation of γ-aminobutyric acid receptors. Deficits in KCC2 activity are implicated in epileptogenesis, but how increased neuronal activity leads to transporter inactivation is ill defined. In vitro, the activity of KCC2 is potentiated via phosphorylation of serine 940 (S940). Here we have examined the role this putative regulatory process plays in determining KCC2 activity during status epilepticus (SE) using knockin mice in which S940 is mutated to an alanine (S940A). In wild-type mice, SE induced by kainate resulted in dephosphorylation of S940 and KCC2 internalization. S940A homozygotes were viable and exhibited comparable basal levels of KCC2 expression and activity relative to WT mice. However, exposure of S940A mice to kainate induced lethality within 30 min of kainate injection and subsequent entrance into SE. We assessed the effect of the S940A mutation in cultured hippocampal neurons to explore the mechanisms underlying this phenotype. Under basal conditions, the mutation had no effect on neuronal Cl(-) extrusion. However, a selective deficit in KCC2 activity was seen in S940A neurons upon transient exposure to glutamate. Significantly, whereas the effects of glutamate on KCC2 function could be ameliorated in WT neurons with agents that enhance S940 phosphorylation, this positive modulation was lost in S940A neurons. Collectively our results suggest that phosphorylation of S940 plays a critical role in potentiating KCC2 activity to limit the development of SE.

γ-Aminobutyric acid type A receptors (GABAARs) are the principal mediators of fast synaptic inhibition in the brain as well as the low persistent extrasynaptic inhibition, both of which are fundamental to proper brain function. Thus unsurprisingly, deficits in GABAARs are implicated in a number of neurological disorders and diseases. The complexity of GABAAR regulation is determined not only by the heterogeneity of these receptors but also by its posttranslational modifications, the foremost, and best characterized of which is phosphorylation. This review will explore the details of this dynamic process, our understanding of which has barely scratched the surface. GABAARs are regulated by a number of kinases and phosphatases, and its phosphorylation plays an important role in governing its trafficking, expression, and interaction partners. Here, we summarize the progress in understanding the role phosphorylation plays in the regulation of GABAARs. This includes how phosphorylation can affect the allosteric modulation of GABAARs, as well as signaling pathways that affect GABAAR phosphorylation. Finally, we discuss the dysregulation of GABAAR phosphorylation and its implication in disease processes.

Mutations that impair the expression and/or function of γ-aminobutyric acid type A (GABAA) receptors can lead to epilepsy. The familial epilepsy γ2(K289M) mutation affects a basic residue conserved in the TM2–3 linker of most GABAA subunits. We investigated the effect on expression and function of the Lys → Met mutation in mouse α1(K278M), β2(K274M), and γ2(K289M) subunits. Compared with cells expressing wild-type and α1β2γ2(K289M) receptors, cells expressing α1(K278M)β2γ2 and α1β2(K274M)γ2 receptors exhibited reduced agonist-evoked current density and reduced GABA potency, with no change in single channel conductance. The low current density of α1β2(K274M)γ2 receptors coincided with reduced surface expression. By contrast the surface expression of α1(K278M)β2γ2 receptors was similar to wild-type and α1β2γ2(K289M) receptors suggesting that the α1(K278M) impairs function. In keeping with this interpretation GABA-activated channels mediated by α1(K278M)β2γ2 receptors had brief open times. To a lesser extent γ2(K289M) also reduced mean open time, whereas β2(K274M) had no effect. We used propofol as an alternative GABAA receptor agonist to test whether the functional deficits of mutant subunits were specific to GABA activation. Propofol was less potent as an activator of α1(K278M)β2γ2 receptors. By contrast, neither β2(K274M) nor γ2(K289M) affected the potency of propofol. The β2(K274M) construct was unique in that it reduced the efficacy of propofol activation relative to GABA. These data suggest that the α1 subunit Lys-278 residue plays a pivotal role in channel gating that is not dependent on occupancy of the GABA binding site. Moreover, the conserved TM2–3 loop lysine has an asymmetric function in different GABAA subunits.

Homomeric 5-hydroxytryptamine type 3A receptors (5-HT3ARs) have a single channel conductance (gamma) below the resolution of single channel recording (966 +/- 75 fS, estimated by variance analysis). By contrast, heteromeric 5-HT3A/B and nicotinic acetylcholine receptors (nAChRs) have picosiemen range gamma values. In this study, single channel recordings revealed that replacement of cytoplasmic membrane-associated (MA) helix arginine 432 (-4'), 436 (0'), and 440 (4') residues by 5-HT3B (-4'Gln, 0'Asp, and 4'Ala) residues increases gamma to 36.5 +/- 1.0 pS. The 0' residue makes the most substantial contribution to gamma of the 5-HT3AR. Replacement of 0'Arg by aspartate, glutamate (alpha7 nAChR subunit MA 0'), or glutamine (beta2 subunit MA 0') increases gamma to the resolvable range (6 pS). By contrast, replacement of 0'Arg by phenylalanine (alpha4 subunit MA 0') reduced gamma to 416 +/- 107 fS. In reciprocal experiments with alpha4beta2 nAChRs (gamma = 31.3 +/- 0.8 pS), replacement of MA 0' residues by arginine in alpha4beta2(Q443R) and alpha4(F588R)beta2 reduced gamma slightly. By contrast, the gamma of double mutant alpha4(F588R)beta2(Q443R) was halved. The MA -4' and 4' residues also influenced gamma of 5-HT3ARs. Replacement of nAChR alpha4 or beta2 MA 4' residues by arginine made current density negligible. By contrast, replacement of both -4' residues by arginine produced functional nAChRs with substantially reduced gamma (11.4 +/- 0.5 pS). Homology models of the 5-HT3A and alpha4beta2 nAChRs against Torpedo nAChR revealed MA -4', 0', and 4' residues within five intracellular portals. This locus may be a common determinant of ion conduction throughout the Cys loop receptor family.

Cognitive dysfunction is a common symptom in many neuropsychiatric disorders and directly correlates with poor patient outcomes. The majority of prolonged inhibitory signaling in the brain is mediated via GABABreceptors (GABABRs), but the molecular function of these receptors in cognition is ill defined. To explore the significance of GABABRs in neuronal activity and cognition, we created mice with enhanced postsynaptic GABABR signaling by mutating the serine 783 in receptor R2 subunit (S783A), which decreased GABABR degradation. Enhanced GABABR activity reduced the expression of immediate-early gene-encoded protein Arc/Arg3.1, effectors that are critical for long-lasting memory. Intriguingly, S783A mice exhibited increased numbers of excitatory synapses and surface AMPA receptors, effects that are consistent with decreased Arc/Arg3.1 expression. These deficits in Arc/Arg3.1 and neuronal morphology lead to a deficit in spatial memory consolidation. Collectively our results suggest a novel and unappreciated role for GABABR activity in determining excitatory neuronal architecture and spatial memory via their ability to regulate Arc/Arg3.1.

The K–Cl cotransporter KCC2 establishes the low intraneuronal Cl – levels required for the hyperpolarizing inhibitory postsynaptic potentials mediated by ionotropic γ-aminobutyric acid receptors (GABA A Rs) and glycine receptors (GlyRs). Decreased KCC2-mediated Cl – extrusion and impaired hyperpolarizing GABA A R- and/or GlyR-mediated currents have been implicated in epilepsy, neuropathic pain, and spasticity. Recent evidence suggests that the intrinsic ion transport rate, cell surface stability, and plasmalemmal trafficking of KCC2 are rapidly and reversibly modulated by the (de)phosphorylation of critical serine, threonine, and tyrosine residues in the C terminus of this protein. Alterations in KCC2 phosphorylation have been associated with impaired KCC2 function in several neurological diseases. Targeting KCC2 phosphorylation directly or indirectly via upstream regulatory kinases might be a novel strategy to modulate GABA- and/or glycinergic signaling for therapeutic benefit.

The K(+) -Cl(-) cotransporter type 2 is the major Cl(-) extrusion mechanism in most adult neurons. This process in turn leads to Cl(-) influx upon activation of γ-aminobutyric acid type A (GABAA ) receptors and the canonical hyperpolarising inhibitory postsynaptic potential. Several neurological disorders are treated with drugs that target and enhance GABAA receptor signaling, including the commonly used benzodiazepine diazepam and the anesthetic propofol. Some of these disorders are also associated with deficits in GABAA signaling and become less sensitive to therapeutic drugs that target GABAA receptors. To date, it is unknown if alterations in the neuronal Cl(-) gradient affect the efficacies of diazepam and propofol. We therefore used the in vitro model of glutamate-induced hyperexcitability to test if alterations in the Cl(-) gradient affect the efficacy of GABAA modulators. We exclusively utilised the gramicidin perforated-patch-clamp configuration to preserve the endogenous Cl(-) gradient in rat neurons. Brief exposure to glutamate reduced the inhibitory efficacy of diazepam within 5 min, which was caused by the collapse of the Cl(-) gradient, and not due to reductions in GABAA receptor number. Unlike diazepam, propofol retained its efficacy by shunting the membrane conductance despite the glutamate-induced appearance of depolarising GABAA -mediated currents. Similarly, pharmacological inhibition of K(+) -Cl(-) cotransporter type 2 by furosemide disrupted Cl(-) homeostasis and reduced the efficacy of diazepam but not propofol. Collectively our results suggest that pathological hyperexcitable conditions could cause the rapid accumulation of intracellular Cl(-) and the appearance of depolarising GABAA -mediated currents that would decrease the efficacy of diazepam.

Picrotoxin and t-butylbicyclophosphorothionate (TBPS) are GABA(A) receptor (GABA(A)R) open channel blockers. However, picrotoxin displaceable [(35)S]TBPS binding to α1β2γ2 GABA(A)Rs occurs in the absence of GABA, suggesting that access to the binding site is independent of activation. Alternatively, spontaneous gating may provide access to the channel. In the absence of episodic GABA application, picrotoxin and TBPS blocked (by 91 ± 3% and 85 ± 5%, respectively) GABA-evoked currents mediated by α1β2γ2 receptors. We used two approaches to inhibit spontaneous GABA(A)R gating, bicuculline, which inhibits spontaneous current in the absence of exogenous agonist and the α1(K278M) mutant subunit. Whole-cell patch-clamp recordings demonstrated that α1(K278M)β2γ2 receptors have negligible spontaneous gating. Application of bicuculline to α1β2γ2 receptors in the absence of exogenous GABA caused a 35% reduction of current blockade by TBPS and reduced [(35)S]TBPS binding by 25%. Consistent with this, in the absence of exogenous GABA, α1(K278M)β2γ2 receptors exhibited reduced blockade by TBPS current compared to wild-type receptors. These data suggest that a decrease in spontaneous gating reduces accessibility of TBPS to its binding site. GABA application during picrotoxin or TBPS administration enhanced α1β2γ2 receptor blockade (to 98% in both cases). The GABA-dependent component of TBPS blockade accounts for the stimulation of [(35)S]TBPS binding to α1β2γ2 receptors seen with GABA (1 μm) application. Moreover, application of GABA at concentrations that cause significant steady-state desensitization reduced [(35)S]TBPS binding. The α1(K278M) subunit slowed desensitization kinetics and increased the rate of deactivation of GABA-evoked currents. Furthermore, there was a marked increase in the GABA EC(50) for desensitization of α1(K278M)β2γ2 receptors associated with a large increase in the GABA-dependent stimulation of [(35)S]TBPS binding. These data establish a relationship between GABA(A)R function and the three phases of [(35)S]TBPS binding seen in the absence and the presence of GABA.

KCC2 is a neuron-specific K(+)-Cl(-) co-transporter that maintains a low intracellular Cl(-) concentration that is essential for hyperpolarizing inhibition mediated by GABA(A) receptors. Deficits in KCC2 activity occur in disease states associated with pathophysiological glutamate release. However, the mechanisms by which elevated glutamate alters KCC2 function are unknown. The phosphorylation of KCC2 residue Ser940 is known to regulate its surface activity. We found that NMDA receptor activity and Ca(2+) influx caused the dephosphorylation of Ser940 in dissociated rat neurons, leading to a loss of KCC2 function that lasted longer than 20 min. Protein phosphatase 1 mediated the dephosphorylation events of Ser940 that coincided with a deficit in hyperpolarizing GABAergic inhibition resulting from the loss of KCC2 activity. Blocking dephosphorylation of Ser940 reduced the glutamate-induced downregulation of KCC2 and substantially improved the maintenance of hyperpolarizing GABAergic inhibition. Reducing the downregulation of KCC2 therefore has therapeutic potential in the treatment of neurological disorders.

Structural models suggest that Arg(436) lies within five cytoplasmic portals of the 5-HT(3A) receptor. We tested both the accessibility of residue 436 and the influence of its charge on single channel conductance (gamma) by substituting Arg(436) with Cys and examining the effect of methanethiosulfonate (MTS) reagents on gamma. Inclusion of positively charged 2-aminoethyl-MTS (MTSEA) within the electrode solution reduced gamma of 5-HT(3A)(R436C) receptors in outside-out patches from 7.8 +/- 0.5 to 5.0 +/- 0.5 picosiemens (pS). To increase gamma, we substituted Arg(436) by Cys in the 5-HT(3A)(R432Q,R440A) mutant, yielding the 5-HT(3A)(QCA) construct with a gamma of 17.7 +/- 0.4 pS. Modification of 5-HT(3A)(QCA) receptors by MTSEA or 2-(trimethylammonium) ethyl-MTS reduced gamma to 8.7 +/- 0.5 and 6.7 +/- 0.4 pS, respectively, both significantly below that of channels exposed to nonpolar propyl-MTS. Extracellular MTSEA, but not 2-(trimethylammonium) ethyl-MTS, crossed the membrane and in so doing slowly (tau = 94 s) reduced gamma. MTSEA more rapidly (tau = 15 s) reduced the gamma of 5-HT(3A)(QCA) receptors in inside-out patches, an effect reversed by the reducing agent dithiothreitol. Cys(436) modification by negatively charged 2-carboxyethyl-MTS and 2-sulfonatoethyl-MTS increasedgamma to 23 +/- 1.0 and 26 +/- 0.7 pS, respectively. MTS reagents did not affect gamma values for 5-HT(3A)(QDA) constructs with Cys substituted for Lys(431) predicted to be outside the entrance to the portals. Collectively, the data demonstrate that the dynamic modification of the charge of a cytoplasmic residue regulates gamma, consistent with the existence of cytoplasmic portals that impose a rate-limiting barrier to ion conduction in Cys loop receptors.

Phasic and tonic inhibitory currents of hippocampal pyramidal neurons exhibit distinct pharmacological properties. Picrotoxin and bicuculline methiodide inhibited both components, consistent with a role for GABAA receptors; however, gabazine, at a concentration that abolished miniature GABAergic inhibitory postsynaptic currents and responses to exogenous GABA, had no effect on tonic currents. Because all GABA-activated GABAA receptors in pyramidal neurons are gabazine-sensitive, it follows that tonic currents are not GABA-activated. Furthermore, picrotoxin-sensitive spontaneous single-channel events recorded from outside-out patches had the same chord conductance as GABA-activated channels and were gabazine-resistant. Therefore, we hypothesize that GABAA receptors, constitutively active in the absence of GABA, mediate tonic current; the failure of gabazine to block tonic current reflects a lack of negative intrinsic efficacy of the antagonist. We compared the negative efficacies of bicuculline and gabazine using the general anesthetic propofol to directly activate GABAA receptors native to pyramidal neurons or alpha1beta3gamma2 receptors recombinantly expressed in human embryonic kidney 293 cells. Propofol activated gabazine-resistant, bicuculline-sensitive currents when applied to either preparation. Although gabazine had negligible efficacy as an inhibitor of propofol-activated currents, it prevented inhibition by bicuculline, which acts as an inverse agonist inhibiting GABA-independent gating. Recombinant alpha1beta1/3gamma2 receptors also mediated agonist-independent tonic currents that were resistant to gabazine and inhibited by bicuculline. Thus, gabazine is a competitive antagonist with negligible negative efficacy and is therefore unable to inhibit GABAA receptors that are active in the absence of GABA because of either anesthetic or spontaneous gating. Moreover, spontaneously active GABAA receptors mediate gabazine-resistant tonic currents in pyramidal neurons.

Nicotinic ACh (acetylcholine) and 5-HT3 (5-hydroxytryptamine type-3) receptors are cation-selective ion channels of the Cys-loop transmitter-gated ion channel superfamily. Numerous lines of evidence indicate that the channel lining domain of such receptors is formed by the alpha-helical M2 domain (second transmembrane domain) contributed by each of five subunits present within the receptor complex. Specific amino acid residues within the M2 domain have accordingly been demonstrated to influence both single-channel conductance (gamma) and ion selectivity. However, it is now clear from work performed on the homomeric 5-HT3A receptor, heteromeric 5-HT3A/5-HT3B receptor and 5-HT3A/5-HT3B receptor subunit chimaeric constructs that an additional major determinant of gamma resides within a cytoplasmic domain of the receptor termed the MA-stretch (membrane-associated stretch). The MA-stretch, within the M3-M4 loop, is not traditionally thought to be implicated in ion permeation and selection. Here, we describe how such observations extend to a representative neuronal nicotinic ACh receptor composed of alpha4 and beta2 subunits and, by inference, probably other members of the Cys-loop family. In addition, we will attempt to interpret our results within the context of a recently developed atomic scale model of the nicotinic ACh receptor of Torpedo marmorata (marbled electric ray).

Mutations that impair the expression and/or function of gamma-aminobutyric acid type A (GABAA) receptors can lead to epilepsy. The familial epilepsy gamma2(K289M) mutation affects a basic residue conserved in the TM2-3 linker of most GABAA subunits. We investigated the effect on expression and function of the Lys --Met mutation in mouse alpha1(K278M), beta2(K274M), and gamma2(K289M) subunits. Compared with cells expressing wild-type and alpha1beta2gamma2(K289M) receptors, cells expressing alpha1(K278M)beta2gamma2 and alpha1beta2(K274M)gamma2 receptors exhibited reduced agonist-evoked current density and reduced GABA potency, with no change in single channel conductance. The low current density of alpha1beta2(K274M)gamma2 receptors coincided with reduced surface expression. By contrast the surface expression of alpha1(K278M)beta2gamma2 receptors was similar to wild-type and alpha1beta2gamma2(K289M) receptors suggesting that the alpha1(K278M) impairs function. In keeping with this interpretation GABA-activated channels mediated by alpha1(K278M)beta2gamma2 receptors had brief open times. To a lesser extent gamma2(K289M) also reduced mean open time, whereas beta2(K274M) had no effect. We used propofol as an alternative GABAA receptor agonist to test whether the functional deficits of mutant subunits were specific to GABA activation. Propofol was less potent as an activator of alpha1(K278M)beta2gamma2 receptors. By contrast, neither beta2(K274M) nor gamma2(K289M) affected the potency of propofol. The beta2(K274M) construct was unique in that it reduced the efficacy of propofol activation relative to GABA. These data suggest that the alpha1 subunit Lys-278 residue plays a pivotal role in channel gating that is not dependent on occupancy of the GABA binding site. Moreover, the conserved TM2-3 loop lysine has an asymmetric function in different GABAA subunits.