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Pesticide action is predominantly measured as a toxicological outcome, with pharmacological impact of sublethal doses on bystander species left largely undocumented. Likewise, chronic exposure, which often results in responses different from acute administration, has also been understudied. In this article, we propose the application of standard pharmacological principles, already used to establish safe clinical dosing regimens in humans, to the 'dosing of the environment'. These principles include relating the steady state dose of an agent to its beneficial effects (e.g. pest control), while minimising harmful impacts (e.g. off-target bioactivity in beneficial insects). We propose the term 'environmental therapeutic window', analogous to that used in mammalian pharmacology, to guide risk assessment. To make pharmacological terms practically useful to environmental protection, quantitative data on pesticide action need to be made available in a freely accessible database, which should include toxicological and pharmacological impacts on both target and off-target species.

There is widespread concern over the global loss of biodiversity. The decline in bee abundances is particularly alarming given their role in pollination; bee losses are a major threat to human food security and ecosystem stability. These losses are associated with intensive land use, which exposes bees to pesticides, particularly neonicotinoids. The latter may harm bees directly and/or exacerbate threats from other chemicals, imported parasites and diseases ( 1 ), or habitat loss. On page 109 of this issue, Mitchell et al. ( 2 ) show that most honeys sampled from around the world between 2012 and 2016 contain neonicotinoids at levels known to be neuroactive in bees. The work highlights the global nature of this threat to bees.

Acute secondary neuronal cell death, as seen in neurodegenerative disease, cerebral ischemia (stroke) and traumatic brain injury (TBI), drives spreading neurotoxicity into surrounding, undamaged, brain areas. This spreading toxicity occurs via two mechanisms, synaptic toxicity through hyperactivity, and excitotoxicity following the accumulation of extracellular glutamate. To date, there are no fast-acting therapeutic tools capable of terminating secondary spreading toxicity within a time frame relevant to the emergency treatment of stroke or TBI patients. Here, using hippocampal neurons (DIV 15–20) cultured in microfluidic devices in order to deliver a localized excitotoxic insult, we replicate secondary spreading toxicity and demonstrate that this process is driven by GluN2B receptors. In addition to the modeling of spreading toxicity, this approach has uncovered a previously unknown, fast acting, GluN2A-dependent neuroprotective signaling mechanism. This mechanism utilizes the innate capacity of surrounding neuronal networks to provide protection against both forms of spreading neuronal toxicity, synaptic hyperactivity and direct glutamate excitotoxicity. Importantly, network neuroprotection against spreading toxicity can be effectively stimulated after an excitotoxic insult has been delivered, and may identify a new therapeutic window to limit brain damage.

This research was funded jointly by BBSRC, DEFRA, NERC, the Scottish Government and The Wellcome Trust, under the Insect Pollinators Initiative (UK) grant BB/1000313/1(CNC). There is growing concern over the risk to bee populations from neonicotinoid insecticides and the long-term consequences of reduced numbers of insect pollinators to essential ecosystem services and food security. Our knowledge of the risk of neonicotinoids to bees is based on studies of imidacloprid and thiamethoxam and these findings are extrapolated to clothianidin based on its higher potency at nicotinic acetylcholine receptors. This study addresses the specificity and consequences of all three neonicotinoids to determine their relative risk to bumblebees at field-relevant levels (2.5 ppb). We find compound-specific effects at all levels (individual cells, bees and whole colonies in semi-field conditions). Imidacloprid and clothianidin display distinct, overlapping, abilities to stimulate Kenyon cells, indicating the potential to differentially influence bumblebee behavior. Bee immobility was induced only by imidacloprid, and an increased vulnerability to clothianidin toxicity only occurred following chronic exposure to clothianidin or thiamethoxam. At the whole colony level, only thiamethoxam altered the sex ratio (more males present) and only clothianidin increased queen production. Finally, both imidacloprid and thiamethoxam caused deficits in colony strength, while no detrimental effects of clothianidin were observed. Given these findings, neonicotinoid risk needs to be considered independently for each compound and target species. Publisher PDF

Graphical abstract, The synthesis, photophysical and biological characterization of a small library of fluorescent 5-HT3 receptor ligands is described. Several of these novel granisetron conjugates have high quantum yields and show high affinity for the human 5-HT3AR.

Although five 5-hydroxytryptamine type 3 (5-HT3) subunits (A-E) have been cloned, knowledge on the regulation of their assembly is limited. RIC-3 has been identified as a chaperone specific for the pentameric ligand-gated nicotinic acetylcholine and 5-HT(3) receptors. Therefore, we examined the impact of RIC-3 on differently composed 5-HT(3) receptors with the focus on 5-HT3C, -D, and -E subunits. The influence of RIC-3 on these receptor subtypes is supported by the presence of RIC3 mRNA in tissues expressing at least one of the subunits 5-HT3C, -D, and -E. Furthermore, immunocytochemical studies on transfected mammalian cells revealed co-localization in the endoplasmic reticulum and direct interaction of RIC-3 with 5-HT3A, -C, -D, and -E. Functional and pharmacological characterization was performed using HEK293 cells expressing 5-HT3A or 5-HT3A + 5-HT3B (or -C, -D, or -E) in the presence or absence of RIC-3. Ca(2+) influx analyses revealed that RIC-3 does not influence the 5-HT concentration-response relationship on 5-HT(3)A receptors but leads to differential increases of 5-HT-induced maximum response (E(max)) on cells expressing different subunits. Increases of E(max) were due to analogously enhanced B(max) values for binding of the 5-HT(3) receptor antagonist [(3)H]GR65630. The observed enhanced cell surface expression of the tested 5-HT3 subunit combinations correlated with the increased surface expression of 5-HT3A as determined by flow cytometry. In conclusion, we showed that RIC-3 can interact with 5-HT3A, -C, -D, and -E subunits and predominantly enhances the surface expression of homomeric 5-HT(3)A receptors in HEK293 cells. These data implicate a possible role of RIC-3 in determining 5-HT(3) receptor composition in vivo.

The hormone leptin can cross the blood–brain barrier and influences numerous brain functions (Harvey, 2007). Indeed, recent studies have demonstrated that leptin regulates activity-dependent synaptic plasticity in the CA1 region of the hippocampus (Shanley et al., 2001; Li et al., 2002; Durakoglugil et al., 2005; Moult et al., 2009). It is well documented that trafficking of AMPA receptors is pivotal for hippocampal synaptic plasticity (Collingridge et al., 2004), but there is limited knowledge of how hormonal systems like leptin influence this process. In this study we have examined how leptin influences AMPA receptor trafficking and in turn how this impacts on excitatory synaptic function. Here we show that leptin preferentially increases the cell surface expression of GluR1 and the synaptic density of GluR2-lacking AMPA receptors in adult hippocampal slices. The leptin-induced increase in surface GluR1 required NMDA receptor activation and was associated with an increase in cytoplasmic PtdIns(3,4,5)P3levels. In addition, leptin enhanced phosphorylation of the lipid phosphatase PTEN which inhibits PTEN function and elevates PtdIns(3,4,5)P3levels. Moreover, inhibition of PTEN mimicked and occluded the effects of leptin on GluR1 trafficking and excitatory synaptic strength. These data indicate that leptin, via a novel pathway involving PTEN inhibition, promotes GluR1 trafficking to hippocampal synapses. This process has important implications for the role of leptin in hippocampal synaptic function in health and disease.

Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system playing critical roles in basal synaptic transmission and mechanisms of learning and memory. Under normal conditions, glutamate is sequestered within synaptic vesicles (~100 mM) with extracellular glutamate concentrations being limited (

The formation of alpha1beta2gamma2epsilon receptors suggests that the epsilon subunit does not displace the single gamma2 subunit in alpha1beta2gamma2 receptors. Thus, epsilon must replace alpha and/or beta subunit(s) if the pentameric receptor structure is to be preserved. To assess the potential for which subunit is replaced in alphabetaepsilon and alphabetagammaepsilon receptors we analyzed the assembly and functional expression of the epsilon subunit with respect to alpha1, beta2 and gamma2 subunits. Using concatenated subunits, we have determined that epsilon is capable of substituting for either (but not both) of the alpha subunits, one of the beta subunits, and possibly the gamma2 subunit. However, the most likely sites at which the epsilon subunit may contribute to receptor function appears to be at position 1 (replaces alpha1) in alphabetagammaepsilon (varepsilon-beta2-alpha1-beta2-gamma2) receptors, or at position 4 (replaces beta2) in alphabetaepsilon (alpha1-beta2-alpha1-varepsilon-beta2) receptors. In both cases, it appears that only a single GABA binding site is present.

RIC-3 has been identified as a chaperone molecule involved in promoting the functional expression of nicotinic acetylcholine and 5-HT(3) receptors in mammalian cells. In this study, we examined the effects of RIC-3a (isoform a) and a truncated isoform (isoform d) on RIC-3 localization, mobility, and aggregation and its effect on 5-HT3 receptor composition in mammalian cells. Human RIC-3a possesses an amino-terminal signal sequence that targets it to the endoplasmic reticulum where it is distributed within the reticular network, often forming large diffuse "slicks" and bright "halo" structures. RIC-3a is highly mobile within and between these compartments. Despite the propensity for RIC-3a to aggregate, its expression enhances the level of surface 5-HT3A (homomeric) receptors. In contrast, RIC-3a exerts an inhibitory action on the surface expression of heteromeric 5-HT3A/B receptors. RIC-3d exhibits an altered subcellular distribution, being localized to the endoplasmic reticulum, large diffuse slicks, tubulo-vesicular structures, and the Golgi. Bidirectional trafficking between the endoplasmic reticulum and Golgi suggests that RIC-3d constitutively cycles between these two compartments. In support of the large coiled-coil domain of RIC-3a being responsible for protein aggregation, RIC-3d, lacking this cytoplasmic domain, does not aggregate or induce the formation of bright aggregates. Regardless of these differences, isoform d is still capable of enhancing homomeric, and inhibiting heteromeric, 5-HT3 receptor expression. Thus, both isoforms of RIC-3 play a role in determining 5-HT3 receptor composition.

Recent studies have implicated the hormone leptin in synaptic plasticity associated with neuronal development and learning and memory. Indeed, leptin facilitates hippocampal long-term potentiation and leptin-insensitive rodents display impaired hippocampal synaptic plasticity suggesting a role for endogenous leptin. Structural changes are also thought to underlie activity-dependent synaptic plasticity and this may be regulated by specific growth factors. As leptin is reported to have neurotrophic actions, we have examined the effects of leptin on the morphology and filopodial outgrowth in hippocampal neurons. Here, we demonstrate that leptin rapidly enhances the motility and density of dendritic filopodia and subsequently increases the density of hippocampal synapses. This process is dependent on the synaptic activation of NR2A-containing NMDA receptors and is mediated by the MAPK (ERK) signaling pathway. As dendritic morphogenesis is associated with activity-dependent changes in synaptic strength, the rapid structural remodeling of dendrites by leptin has important implications for its role in regulating hippocampal synaptic plasticity and neuronal development.

N-terminally tagged CB1 receptor fusion proteins, incorporating enhanced green fluorescent protein (GFP) or super-ecliptic pHluorin (SEP), were generated to study CB1 receptor trafficking and cell surface receptor expression in live COS7 and HEK293 cells and hippocampal neurons. An artificial signal sequence (SS) was required for efficient surface expression of CB1 receptor chimeras, which behaved like wild-type CB1 receptors in functional assays. Treatment with cannabinoid ligands led to a rapid down-regulation of SS-GFP-CB1 from the plasma membrane in COS7 and HEK293 cells, associated with trafficking into cytosolic vesicles. Activation of CB1 receptors was also linked with a time-dependent reduction in cell surface SEP-CB1 fluorescence and incorporation of the construct into acidic endosomes, revealed following exposure to NH4Cl. In live hippocampal neurons, SEP-CB1 fluorescence was largely restricted to the axon, consistent with its polarised surface expression. Thus, these new molecular tools are well suited for studying CB1 receptor trafficking and a new generation of GPCR chimeras incorporating SEP at the N-terminus will be especially useful for monitoring dynamic changes in cell surface receptor expression in living cells.

In central neurons, the cell-surface distribution of cannabinoid receptor subtype-1 (CB(1)) is highly polarized toward axons and is associated with synaptic terminals, in which it is well-positioned to modulate neurotransmitter release. It has been suggested that high levels of constitutive activity mediate CB(1) receptor axonal targeting, leading to domain-specific endocytosis. We have investigated further the mechanisms that underlie CB(1) receptor axonal polarization in hippocampal neurons and found that constitutive activity is not an essential requirement for this process. We demonstrate that the cell-surface distribution of an N-terminally tagged, fluorescent CB(1) receptor fusion-protein is almost exclusively localized to the axon when expressed in cultured hippocampal neurons. Inhibition of endocytosis by cotransfection with a dominant-negative dynamin-1 (K44A) mutant traps both recombinant and endogenous CB(1) receptors at the somatodendritic cell surface. However, this effect could not be mimicked by inhibiting constitutive activity or receptor activation, either by expressing mutant receptors that lack these properties or by treatment with CB(1) receptor antagonists possessing inverse agonist activity. These data are consistent with a revised model in which domain-specific endocytosis regulates the functional polarization of CB(1) receptors, but this process is distinct from constitutive activity.

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.

Background: Altered levels of 5-HT are thought to contribute to cognitive and gastrointestinal dysfunction. Results: Intracellular accumulation of 5-HT leads to a prolonged loss of 5-HT3 receptor function. Conclusion: 5-HT3 receptor desensitization is maintained by a chronic release of 5-HT. Significance: Prolonged 5-HT3 receptor dysfunction may result from the clinical use of selective serotonin reuptake inhibitors or pathologically raised 5-HT levels., The serotonergic synapse is dynamically regulated by serotonin (5-hydroxytryptamine (5-HT)) with elevated levels leading to the down-regulation of the serotonin transporter and a variety of 5-HT receptors, including the 5-HT type-3 (5-HT3) receptors. We report that recombinantly expressed 5-HT3 receptor binding sites are reduced by chronic exposure to 5-HT (IC50 of 154.0 ± 45.7 μm, t½ = 28.6 min). This is confirmed for 5-HT3 receptor-induced contractions in the guinea pig ileum, which are down-regulated after chronic, but not acute, exposure to 5-HT. The loss of receptor function does not involve endocytosis, and surface receptor levels are unaltered. The rate and extent of down-regulation is potentiated by serotonin transporter function (IC50 of 2.3 ± 1.0 μm, t½ = 3.4 min). Interestingly, the level of 5-HT uptake correlates with the extent of down-regulation. Using TX-114 extraction, we find that accumulated 5-HT remains soluble and not membrane-bound. This cytoplasmically sequestered 5-HT is readily releasable from both COS-7 cells and the guinea pig ileum. Moreover, the 5-HT level released is sufficient to prevent recovery from receptor desensitization in the guinea pig ileum. Together, these findings suggest the existence of a novel mechanism of down-regulation where the chronic release of sequestered 5-HT prolongs receptor desensitization.

RIC-3 has been identified as a molecule essential for the recruitment of functional nicotinic acetylcholine receptors composed of alpha7, but it exhibits inhibitory effects on alpha4beta2 or alpha3beta4 receptors. In this study, we investigated the role of RIC-3 in the recruitment of 5-hydroxytryptamine type 3A (5-HT(3A)) receptors to the cell surface. Although RIC-3 is not essential for the surface transport of 5-HT(3A) receptors, we found that its presence enhances both receptor transport and function in a concentration-dependent manner. RIC-3 is localized to the endoplasmic reticulum, as evidenced by co-localization with the chaperone molecule, binding protein (BiP). RIC-3 is not detected at significant levels on the cell surface when expressed alone or in the presence of 5-HT(3A). RIC-3 and 5-HT(3A) show a low level interaction that is transient (4 h). That RIC-3 can interact with an endoplasmic reticulum-retained 5-HT(3A) construct, combined with the transient interaction observed and lack of significant surface-expressed RIC-3, suggests that RIC-3 may play a role in 5-HT(3A) receptor folding, assembly, or transport to the cell surface.

We have investigated the role of Ca2+ accumulation and neuronal injury in cerebellar granule neurons after glutamate receptor overactivation. After the removal of the free cytosolic Ca2+ we identified an extensive second Ca2+ fraction (SCF) that is retained within the neurons after glutamate receptor overactivation. The SCF reaches a plateau within 10 min with the magnitude of this SCF accumulation reflecting the extent of the neuronal injury that occurs within the neurons. The existence of this SCF is sensitive to both NMDA receptor antagonists and mitochondrial inhibitors but is unaffected by agents that deplete endoplasmic reticulum Ca2+, indicating that this Ca2+ fraction may be located within the mitochondria. Through the isolation of mitochondria from cerebellar granule neurons treated with glutamate we have shown that the majority of the SCF is mitochondrial in location. On the removal of the glutamate stimulus the SCF recovers at a slower rate than the free Ca2+ concentration within the neuron. This is intriguing, as it implies a capacity to remember previous excitatory events. Most significantly we have shown that a short pre-application of subthreshold glutamate or kainate blocks both SCF Ca2+ accumulation and extensive neuronal injury in response to high concentrations of glutamate. These findings may be relevant to the observations of pre-conditioning in the brain and heart.

Mammalian gamma-aminobutyric acid type A (GABAA) receptors are constructed from a large repertoire of subunits (alpha1-alpha6, beta1-beta3, gamma1-gamma3, delta, epsilon, theta and pi) into a pentameric ion channel. GABA(A) receptor assembly occurs within the endoplasmic reticulum (ER) and involves interactions with chaperone molecules. Only specific subunit combinations can produce functional surface receptors (with a fixed stoichiometry); other subunit combinations are retained within the ER and degraded. Thus, receptor assembly occurs by defined pathways to limit the diversity of GABAA receptors. The key to understanding how receptor diversity is achieved and controlled is the identification of assembly signals capable of distinguishing between other subunit partners. Analysis of an assembly box in alpha1 (residues 57-68) has revealed an absolute requirement for this region in the assembly of alphabeta receptors. Furthermore, a selective requirement for a single amino acid (R66) is observed for the assembly of alpha1beta2, but not alpha1beta1 or alpha1beta3, receptors. In addition, we have characterized an assembly signal in the beta3 subunit that is capable of driving the assembly of beta3, gamma2beta3 and alpha1beta3 receptors. Interestingly, this signal does not appear to utilize the alpha1 assembly box, suggesting the presence of alternative assembly signals within the alpha1 subunit. Although this beta3 signal is sufficient to permit the formation of betagamma receptors it is not necessary, suggesting that alternative assembly signals also exist within the beta3 subunit. These findings support the belief that GABAA receptor assembly occurs via multiple defined pathways that may be determined by subunit availability.

Two subunits of the 5-hydroxytryptamine type 3 (5-HT3) have been identified (5-HT3A and 5-HT3B) that assemble into homomeric (5-HT3A) and heteromeric (5-HT3A+5-HT3B) complexes. Unassembled 5-HT3B subunits are efficiently retained within the cell. In this study, we address the mechanism controlling the release of 5-HT3B from the endoplasmic reticulum (ER). An analysis of chimeric 5-HT3A receptor(R).5-HT3BR constructs suggests the presence of elements downstream of the first transmembrane domain of 5-HT3B subunits that inhibit cell surface expression. To investigate this possibility, truncated 5-HT3B subunits were constructed and assessed for their ability to access the cell surface in COS-7 and ts201 cells. Using this approach, we have identified the presence of an ER retention signal located within the first cytoplasmic loop between transmembrane domains I and II of 5-HT3B. Transplantation of this signal (CRAR) into the homologous region of 5-HT3A results in the ER retention of this subunit until rescued by co-assembly with wild-type 5-HT3A. The mutation of this ER retention signal in 5-HT3B (5-HT3BSGER) does not lead to cell surface expression, suggesting the presence of other signals or mechanisms to control the surface expression of 5-HT3BRs. The generation of truncated 5-HT3BSGER constructs confirmed that the CRAR signal does play an important role in the ER retention of 5-HT3B.

Type A gamma -aminobutyric acid receptors (GABA(A)), the major sites of fast synaptic inhibition in the brain, ave believed to be predominantly composed of alpha, beta, and gamma subunits. To examine the membrane trafficking of GABA(A) receptors we have produced gamma 2L subunit chimeras with green fluorescent protein (GFP), Addition of GFP to the N-terminus of the gamma2 subunit (gamma 2L-GFPN) was functionally silent for alpha1 beta2 gamma 2L-GFPN receptors expressed in A293 cells. Furthermore, this chimera allowed the visualization of receptor membrane targeting and endocytosis in live cells. In contrast, incorporation of GFP at the C-terminus reduced subunit stability, impairing assembly with receptor alpha and beta subunits. Using gamma 2L-GFPN we were able to demonstrate that targeting of the gamma2 subunit to GABAergic synapses in hippocampal neurons was dependent upon coassembly with receptor alpha and beta subunits. Together our results demonstrate that the assembly and membrane targeting of GABA(A) receptors composed of alpha1 beta2 gamma 2L-GFPN subunits follow similar itineraries in heterologous systems and neurons.

GABA(A) receptors can be constructed from a range of differing subunit isoforms: alpha, beta, gamma, delta, and epsilon. Expression studies have revealed that production of GABA-gated channels is achieved after coexpression of alpha and beta subunits. The expression of a gamma subunit isoform is essential to confer benzodiazepine sensitivity on the expressed receptor. However, how the specificity of subunit interactions is controlled during receptor assembly remains unknown. Here we demonstrate that residues 58-67 within alpha subunit isoforms are important in the assembly of receptors comprised of alphabeta and alphabetagamma subunits. Deletion of these residues from the alpha1 or alpha6 subunits results in retention of either alpha subunit isoform in the endoplasmic reticulum on coexpression with the beta3, or beta3 and gamma2 subunits. Immunoprecipitation revealed that residues 58-67 mediated oligomerization of the alpha1 and beta3 subunits, but were without affect on the production of alpha/gamma complexes. Within this domain, glutamine 67 was of central importance in mediating the production of functional alpha1beta3 receptors. Mutation of this residue resulted in a drastic decrease in the cell surface expression of alpha1beta3 receptors and the resulting expression of beta3 homomers. Sucrose density gradient centrifugation revealed that this residue was important for the production of a 9S alpha1beta3 complex representing functional GABA(A) receptors. Therefore, our studies detail residues that specify GABA(A) receptor alphabeta subunit interactions. This domain, which is conserved in all alpha subunit isoforms, will therefore play a critical role in the assembly of GABA(A) receptors composed of alphabeta and alphabetagamma subunits.

Type A gamma-aminobutyric acid receptors (GABA(A)), the major sites of fast synaptic inhibition in the brain, are believed to be composed predominantly of alpha, beta, and gamma subunits. Although cell surface expression is essential for GABA(A) receptor function, little is known regarding its regulation. To address this issue, the membrane stability of recombinant alpha(1)beta(2) or alpha(1)beta(2)gamma(2) receptors was analyzed in human embryonic kidney cells. Alpha(1)beta(2)gamma(2) but not alpha(1)beta(2) receptors were found to recycle constitutively between the cell surface and a microtubule-dependent, perinuclear endosomal compartment. Similar GABA(A) receptor endocytosis was also seen in cultured hippocampal and cortical neurons. GABA(A) receptor surface levels were reduced upon protein kinase C (PKC) activation. Like basal endocytosis, this response required the gamma(2) subunit but not receptor phosphorylation. Although inhibiting PKC activity did not block alpha(1)beta(2)gamma(2) receptor endocytosis, it did prevent receptor down-regulation, suggesting that PKC activity may block alpha(1)beta(2)gamma(2) receptor recycling to the cell surface. In agreement with this observation, blocking recycling from endosomes with wortmannin selectively reduced surface levels of gamma(2)-containing receptors. Together, our results demonstrate that the surface stability of GABA(A) receptors can be dynamically and specifically regulated, enabling neurons to modulate cell surface receptor number upon the appropriate cues.

GABA(A) receptors are believed to be heteropentamers that can be constructed from six subunit classes: alpha(1-6), beta(1-4), gamma(1-3), delta, epsilon, and pi. Given that individual neurons often express multiple receptor subunits, it is important to understand how these receptors assemble. To determine which domains of receptor subunits control assembly we have exploited the differing capabilities of the beta 2 and beta 3 subunits to form functional cell surface homomeric receptors. Using a chimeric approach, we have identified four amino acids in the N-terminal domain of the beta 3 subtlnit that mediate functional cell surface expression of this subunit compared with beta 2, which is retained within the endoplasmic reticulum. Substitution of these four amino acids-glycine 171, lysine 173, glutamate 179, and arginine 180-into the beta 2 subunit was sufficient to enable the beta 2 subunit to homo-oligomerize. The effect of this putative "assembly signal" on the production of heteromeric receptors composed of crp and pr subunits was also analyzed. This signal was not critical for the formation of receptors composed of either alpha 1 beta 2 or alpha 1 beta 3 subunits, suggesting that mutation of these residues did not disrupt subunit folding. However, this signal was important in the formation of beta gamma 2 receptors. These residues did not seem to affect the initial association of beta 2 and gamma 2 subunits but appeared to be important for the subsequent production of functional receptors. Our studies identify, for the first time, key residues within the N-terminal domains of receptor beta subunits that mediate the selective assembly of GABA(A) receptors.

1. The functional role played by the postulated disulphide bridge in gamma-aminobutyric acid type A (GABAA) receptors and its susceptibility to oxidation and reduction were studied using recombinant (murine receptor subunits expressed in human embryonic kidney cells) and rat neuronal GABAA receptors in conjunction with whole-cell and single channel patch-clamp techniques. 2. The reducing agent dithiothreitol (DTT) reversibly potentiated GABA-activated responses (IGABA) of alpha1beta1 or alpha1beta2 receptors while the oxidizing reagent 5, 5'-dithio-bis-(2-nitrobenzoic acid) (DTNB) caused inhibition. Redox modulation of IGABA was independent of GABA concentration, membrane potential and the receptor agonist and did not affect the GABA EC50 or Hill coefficient. The endogenous antioxidant reduced glutathione (GSH) also potentiated IGABA in alpha1beta2 receptors, while both the oxidized form of DTT and glutathione (GSSG) caused small inhibitory effects. 3. Recombinant receptors composed of alpha1beta1gamma2S or alpha1beta2gamma2S were considerably less sensitive to DTT and DTNB. 4. For neuronal GABAA receptors, IGABA was enhanced by flurazepam and relatively unaffected by redox reagents. However, in cultured sympathetic neurones, nicotinic acetylcholine-activated responses were inhibited by DTT whilst in cerebellar granule neurones, NMDA-activated currents were potentiated by DTT and inhibited by DTNB. 5. Single GABA-activated ion channel currents exhibited a conductance of 16 pS for alpha1beta1 constructs. DTT did not affect the conductance or individual open time constants determined from dwell time histograms, but increased the mean open time by affecting the channel open probability without increasing the number of cell surface receptors. 6. A kinetic model of the effects of DTT and DTNB suggested that the receptor existed in equilibrium between oxidized and reduced forms. DTT increased the rate of entry into reduced receptor forms and also into desensitized states. DTNB reversed these kinetic effects. 7. Our results indicate that GABAA receptors formed by alpha and beta subunits are susceptible to regulation by redox agents. Inclusion of the gamma2 subunit in the receptor, or recording from some neuronal GABAA receptors, resulted in reduced sensitivity to DTT and DTNB. Given the suggested existence of alphabeta subunit complexes in some areas of the central nervous system together with the generation and release of endogenous redox compounds, native GABAA receptors may be subject to regulation by redox mechanisms.

A key new risk to our pollinators has been identified as exposure to neonicotinoid insecticides. These discoveries have refuelled the debate over whether or not the neonicotinoid insecticides should be banned and conflicting evidence is used in this battle. However, the issue is not black or white, but gray. It is not an issue of whether the neonicotinoids are toxic to insects or not. Clearly, all insecticides were designed and optimized for this attribute. The real question is, or at least should be, which insecticide is the safest for use for a particular need.

Pesticides that target cholinergic neurotransmission are highly effective, but their use has been implicated in insect pollinator population decline. Honeybees are exposed to two widely used classes of cholinergic pesticide: neonicotinoids (nicotinic receptor agonists) and organophosphate miticides (acetylcholinesterase inhibitors). Although sublethal levels of neonicotinoids are known to disrupt honeybee learning and behaviour, the neurophysiological basis of these effects has not been shown. Here, using recordings from mushroom body Kenyon cells in acutely isolated honeybee brain, we show that the neonicotinoids imidacloprid and clothianidin, and the organophosphate miticide coumaphos oxon, cause a depolarization-block of neuronal firing and inhibit nicotinic responses. These effects are observed at concentrations that are encountered by foraging honeybees and within the hive, and are additive with combined application. Our findings demonstrate a neuronal mechanism that may account for the cognitive impairments caused by neonicotinoids, and predict that exposure to multiple pesticides that target cholinergic signalling will cause enhanced toxicity to pollinators., Exposure to pesticides can disrupt foraging and navigation behaviour in bees. Palmer et al. use electrophysiology to show that two neonicotinoids and an organophosphate miticide cause neuronal dysfunction in the honeybee brain at environmentally relevant concentrations.

Cholinergic signalling is fundamental to neuro-muscular function in most organisms. Sub-lethal doses of neurotoxic pesticides that target cholinergic signalling can alter the behaviour of insects in subtle ways; their influence on non-target organisms may not be readily apparent in simple mortality studies. Beneficial arthropods such as honeybees perform sophisticated behavioural sequences during foraging that, if influenced by pesticides, could impair foraging success and reduce colony health. Here, we investigate the behavioural effects on honeybees of exposure to a selection of pesticides that target cholinergic signalling by inhibiting acetylcholinesterase (AChE). To examine how continued exposure to AChE inhibitors affected motor function, we fed adult foraging worker honeybees sub-lethal concentrations of these compounds in sucrose solution for 24 h. Using an assay for locomotion in bees, we scored walking, stopped, grooming, and upside down behaviour continuously for 15 min. At a 10nM concentration, all the AChE inhibitors caused similar effects on behaviour, notably increased grooming activity and changes in the frequency of bouts of behaviour such as head grooming. Coumaphos caused dose-dependent effects on locomotion as well as grooming behaviour, and a 1µM concentration of coumaphos induced symptoms of malaise such as abdomen grooming and defecation. Biochemical assays confirmed that the 4 compounds we assayed (coumaphos, aldicarb, chlorpyrifos, and donepezil) or their metabolites acted as AChE inhibitors in bees. Furthermore, we show that transcript expression levels of two honeybee acetylcholinesterase inhibitors were selectively upregulated in the brain and in gut tissues in response to AChE inhibitor exposure. The results of our study imply that the effects of pesticides that rely on this mode of action have subtle yet profound effects on physiological effects on behaviour that could lead to reduced survival.

1. In response to evidence of insect pollinator declines, organisations in many sectors, including the food and farming industry, are investing in pollinator conservation. They are keen to ensure that their efforts use the best available science. 2. We convened a group of 32 ‘conservation practitioners’ with an active interest in pollinators and 16 insect pollinator scientists. The conservation practitioners include representatives from UK industry (including retail), environmental non-government organisations and nature conservation agencies. 3. We collaboratively developed a long list of 246 knowledge needs relating to conservation of wild insect pollinators in the UK. We refined and selected the most important knowledge needs, through a three-stage process of voting and scoring, including discussions of each need at a workshop. 4. We present the top 35 knowledge needs as scored by conservation practitioners or scientists. We find general agreement in priorities identified by these two groups. The priority knowledge needs will structure ongoing work to make science accessible to practitioners, and help to guide future science policy and funding. 5. Understanding the economic benefits of crop pollination, basic pollinator ecology and impacts of pesticides on wild pollinators emerge strongly as priorities, as well as a need to monitor floral resources in the landscape.

gamma-aminobutyric acid type A (GABAA) receptors are the major sites of fast synaptic inhibition in the brain. They are constructed from four subunit classes with multiple members: alpha (1-6), beta (1-4), gamma (1-4), and delta (1). The contribution of subunit diversity in determining receptor subcellular targeting was examined in polarized Madin-Darby canine kidney (MDCK) cells. Significant detection of cell surface homomeric receptor expression by a combination of both immunological and electrophysiological methodologies was only found for the beta 3 subunit. Expression of alpha/beta binary combinations resulted in a nonpolarized distribution for alpha 1 beta 1 complexes, but specific basolateral targeting of both alpha 1 beta 2 and alpha 1 beta 3 complexes. The polarized distribution of these alpha/beta complexes was unaffected by the presence of the gamma 2S subunit. Interestingly, delivery of receptors containing the beta 3 subunit to the basolateral domain occurs via the apical surface. These results show that beta subunits can selectively target GABAA receptors to distinct cellular locations. Changes in the spatial and temporal expression of beta-subunit isoforms may therefore provide a mechanism for relocating GABAA receptor function between distinct neuronal domains. Given the critical role of these receptors in mediating synaptic inhibition, the contribution of different beta subunits in GABAA receptor function, may have implications in neuronal development and for receptor localization/clustering.

We have developed a novel technique with which to investigate the morphological basis of exocytotic traffic. We have used expression of HRP from cDNA in a variety of cells in combination with peroxidase cytochemistry to outline traffic into and out of the Golgi apparatus at the electron microscopic level with very high sensitivity. A secretory form of the peroxidase (ssHRP) is active from the beginning of the secretory pathway and the activity is efficiently cleared from cells. Investigation of the morphological elements involved in the itinerary of soluble ER proteins using ssHRP tagged with the ER retention motif (ssHRPKDEL) shows that it progresses through the Golgi stack no further than the cis-most element. Traffic between the RER and the Golgi stack as outlined by ssHRPKDEL occurs via vesicular carriers as well as by tubular elements. ssHRP has also been used to investigate the trans side of the Golgi complex, where incubation at reduced temperatures outlines the trans-Golgi network with HRP reaction product. Tracing the endosomal compartment with transferrin receptor in double-labeling experiments with ssHRP fails to show any overlap between these two compartments.

The 5-HT(3) and GABA(A) receptors are members of the Cys-loop family of neurotransmitter-gated ion channels that also include receptors for glycine and acetylcholine. The 5-HT(3) and acetylcholine receptors (cationic ion channels) and the GABA(A) and glycine receptors (anionic ion channels) generally depolarize or hyperpolarize, respectively, the neuronal membrane. Within the amino-terminal extracellular region, all members of this family exhibit a similar architecture of ligand binding domains and a number of key residues are completely conserved. The molecular characterization of their ligand binding and gating characteristics has benefited from the existence of a large repertoire of individual subunits that contribute to the pentameric ion channel. Although differences do exist, advances in our knowledge of one member offers valuable insight into the family as a whole. Each member of the Cys-loop receptors (and all other multimeric ion channels) must face the same challenges: How to assemble individual subunits into an ion channel and which subunits to use? How are assembled receptors distinguished from those that are unassembled or misassembled, then exported from the endoplasmic reticulum and delivered to the cell surface? How are they targeted to, and anchored at synaptic and extrasynaptic sites? How and when are they to be removed from these sites to provide long-term regulation of neuronal activity? In this review, we summarize our current knowledge for the 5-HT(3) and GABA(A) receptors that have provided complementary information and helped us build an overall picture of how receptor biogenesis and trafficking occurs.

Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system (CNS) and is normally stored intracellularly. However, in instances of CNS injury or disease, increased concentrations of extracellular glutamate can result in the over-activation of ionotropic glutamate receptors and trigger neuronal cell death (termed excitotoxicity). Two early hallmarks of such neuronal toxicity are mitochondrial dysfunction (depolarisation, decreased ATP synthesis, structural collapse and potential opening of the permeability transition pore) and the formation of focal swellings (also termed varicosities/beads) along the length of the dendrites. In this review, we summarise current knowledge of the mechanisms that underlie these early excitotoxic events as well as the mechanisms that facilitate dendritic recovery following termination of the excitotoxic insult.

Mitochondrial dysfunction (depolarization and structural collapse), cytosolic ATP depletion, and neuritic beading are early hallmarks of neuronal toxicity induced in a variety of pathological conditions. We show that, following global exposure to glutamate, mitochondrial changes are spatially and temporally coincident with dendritic bead formation. During oxygen-glucose deprivation, mitochondrial depolarization precedes mitochondrial collapse, which in turn is followed by dendritic beading. These events travel as a wave of activity from distal dendrites toward the neuronal cell body. Despite the spatiotemporal relationship between dysfunctional mitochondria and dendritic beads, mitochondrial depolarization and cytoplasmic ATP depletion do not trigger these events. However, mitochondrial dysfunction increases neuronal vulnerability to these morphological changes during normal physiological activity. Our findings support a mechanism whereby, during glutamate excitotoxicity, Ca(2+) influx leads to mitochondrial depolarization, whereas Na(+) influx leads to an unsustainable increase in ATP demand (Na(+),K(+)-ATPase activity). This leads to a drop in ATP levels, an accumulation of intracellular Na(+) ions, and the subsequent influx of water, leading to microtubule depolymerization, mitochondrial collapse, and dendritic beading. Following the removal of a glutamate challenge, dendritic recovery is dependent upon the integrity of the mitochondrial membrane potential, but not on a resumption of ATP synthesis or Na(+),K(+)-ATPase activity. Thus, dendritic recovery is not a passive reversal of the events that induce dendritic beading. These findings suggest that the degree of calcium influx and mitochondrial depolarization inflicted by a neurotoxic challenge, determines the ability of the neuron to recover its normal morphology.

Background The serotonin [5-hydroxytryptamine (5-HT)]-gated ion channel 5.HT 3 is involved in the mediation of postoperative and radiotherapy/chemotherapy-induced nausea/emesis and in irritable bowel syndrome. It has also been suggested to play a role in various psychiatric diseases. Five naturally occurring single nucleotide polymorphisms leading to amino acid changes have been identified in the human 5.HT 3A gene. Methods and results We investigated the functional effects of these polymorphisms on the 5.HT 3A receptor using fluorescence-based cellular assays. Notably, variants A33T, S253N, and M2571 displayed 5-HT-induced maximal responses of 3-64% of the wild-type response, whereas R344H and P391 R exhibited wild-type-like function. All variants displayed wild-type-like potencies of 5-HT and three 5.HT 3 antagonists. Furthermore, all variants displayed K d values similar to that of the wild-type receptor in a [ 3 H]GR65630-binding assay. The surface expression of A33T, M257I, and R344H was reduced 2-4-fold compared with the wild-type, despite similar total expression levels. Finally, coexpression of wild-type 5.HT 3A or 5.HT 3B subunits with 5.HT 3A variants A33T, S253N, or M257I resulted in mixed or heteromeric receptors, characterized by significantly reduced maximal responses to 5-HT compared with the wild-type receptors. Conclusions Three polymorphisms of the 5-HT 3A gene gave rise to functionally impaired receptors whose function could not be rescued by either wild-type 5-HT 3A or 5-HT 3B . Three of the variant receptors were surface-expressed at reduced levels in spite of total expression levels similar to wild-type, indicating that these variants affect receptor biogenesis and/or trafficking. These severe single nucleotide polymorphism effects hold promise for identification of new 5-HT 3A gene-disease causalities.

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.

We have investigated the role of Ca2+ accumulation and neuronal injury in cerebellar granule neurons after glutamate receptor overactivation. After the removal of the free cytosolic Ca2+ we identified an extensive second Ca2+ fraction (SCF) that is retained within the neurons after glutamate receptor overactivation. The SCF reaches a plateau within 10 min with the magnitude of this SCF accumulation reflecting the extent of the neuronal injury that occurs within the neurons. The existence of this SCF is sensitive to both NMDA receptor antagonists and mitochondrial inhibitors but is unaffected by agents that deplete endoplasmic reticulum Ca2+, indicating that this Ca2+ fraction may be located within the mitochondria. Through the isolation of mitochondria from cerebellar granule neurons treated with glutamate we have shown that the majority of the SCF is mitochondrial in location. On the removal of the glutamate stimulus the SCF recovers at a slower rate than the free Ca2+ concentration within the neuron. This is intriguing, as it implies a capacity to remember previous excitatory events. Most significantly we have shown that a short pre-application of subthreshold glutamate or kainate blocks both SCF Ca2+ accumulation and extensive neuronal injury in response to high concentrations of glutamate. These findings may be relevant to the observations of pre-conditioning in the brain and heart.

Given the association of a gamma2 mutation (R43Q) with epilepsy and the reduced cell surface expression of mutant receptors, we investigated a role for this residue in alpha1beta2gamma2 receptor assembly when present in each subunit. Regardless of which subunit contained the mutation, mutant GABA(A) receptors assembled poorly into functional cell surface receptors. The low level of functional expression gives rise to reduced GABA EC50s (alpha1(R43Q)beta2gamma2 and alpha1beta2(R43Q)gamma2) or reduced benzodiazepine potentiation of GABA-evoked currents (alpha1beta2gamma2(R43Q)). We determined that a 15-residue peptide surrounding R43 is capable of subunit binding, with a profile that reflected the orientation of subunits in the pentameric receptor. Subunit binding is perturbed when the R43Q mutation is present suggesting that this residue is critical for the formation of inter-subunit contacts at (+) interfaces of GABAA subunits. Rather than being excluded from receptors, gamma2(R43Q) may form non-productive subunit interactions leading to a dominant negative effect on other receptor subtypes.

Elevated glycogen synthase kinase-3 (GSK-3) activity is associated with Alzheimer disease. We have found that collapsin response mediator proteins (CRMP) 2 and 4 are physiological substrates of GSK-3. The amino acids targeted by GSK-3 comprise a hyperphosphorylated epitope first identified in plaques isolated from Alzheimer brain. Expression of wild type CRMP2 in primary hippocampal neurons or SH-SY5Y neuroblastoma cells promotes axon elongation. However, a GSK-3-insensitive CRMP2 mutant has dramatically reduced ability to promote axon elongation, a similar effect to pharmacological inhibition of GSK-3. Hence, we propose that phosphorylation of CRMP proteins by GSK-3 regulates axon elongation. This work provides a direct connection between hyperphosphorylation of these residues and elevated GSK-3 activity, both of which are observed in Alzheimer brain.

The Cys-loop receptors constitute an important superfamily of LGICs (ligand-gated ion channels) comprising receptors for acetylcholine, 5-HT3 (5-hydroxytryptamine; 5-HT3 receptors), glycine and GABA (γ -aminobutyric acid; GABAA receptors). A vast knowledge of the structure of the Cys-loop superfamily and its impact on channel function have been accrued over the last few years, leading to exciting new proposals on how ion channels open and close in response to agonist binding. Channel opening is initiated by the extracellular association of agonists to discrete binding pockets, leading to dramatic conformational changes, culminating in the opening of a central ion pore. The importance of channel structure is exemplified in the allosteric modulation of channel function by the binding of other molecules to distinct sites on the channel, which exerts an additional level of control on their function. The subsequent conformational changes (gating) lead to channel opening and ion transport. Following channel pore opening, ion selectivity is determined by receptor structure in, and around, the ion pore. As a final level of control, cytoplasmic determinants control the magnitude (conductance) of ion flow into the cell. Thus the Cys-loop receptors are complex molecular motors, with moving parts, which can transduce extracellular signals across the plasma membrane. Once the full mechanical motions involved are understood, it may be possible to design sophisticated therapeutic agents to modulate their activity, or at least be able to throw a molecular spanner into the works! Despite the large number of membrane proteins, the highresolution structure of only a handful of mammalian membrane proteins has been elucidated. The major barriers, to what has been a determined effort, lie in the difficulties of obtaining sufficient amounts of pure protein and crystallization for X-ray diffraction studies. The first problem has been side-stepped by the investigation of bacterial proteins or atypical circumstances in which the protein is naturally concentrated, such as for the acetylcholine

I agree that all valuable input should be acknowledged by authorship, from technical to inspirational. The issue is the inclusion of undeserving authors that manipulate the system to claim authorship and the more generous situation of helping someone along in their career. Perhaps a situation in which all authors are assigned a percentage for their contribution would be more appropriate (and quantitative). Authors providing the funding to support the research may remove their contribution and authorship if an amicable distribution of credit is not agreed. With respect to referees, we all have shoddy feedback that is unsubstantiated (or worse). It is time that referees are paid for their professional services, but at the same time, as these are professional assessments, I do not see the justification for maintaining their anonymity. However, I do see very good reason for keeping the anonymity of the authors and their institute until the reviewing procedure is complete. This is entirely irrelevant, or it should be! What can be fairer than a paid professional assessment of work conducted by unknown scientists and the credit for the work assigned by the authors?

Key to understanding how receptor diversity is achieved and controlled is the identification of selective assembly signals capable of distinguishing between other subunit partners. We have identified that the beta1-3 subunits exhibit distinct assembly capabilities with the gamma2L subunit. Similarly, analysis of an assembly box in alpha1-(57-68) has revealed an absolute requirement for this region in the assembly of alphabeta receptors. Furthermore, a selective requirement for a single amino acid (Arg-66), previously shown to be essential for the formation of the low affinity GABA binding site, is observed. This residue is critical for the assembly of alpha1beta2 but not alpha1beta1 or alpha1beta3 receptors. We have confirmed the ability of the previously identified GKER signal in beta3 to direct the assembly of betagamma receptors. The GKER signal is also involved in driving assembly with the alpha1 subunit, conferring the ability to assemble with alpha1(R66A) on the beta2 subunit. Although this signal is sufficient to permit the formation of beta2gamma2 receptors, it is not necessary for beta3gamma2 receptor formation, suggesting the existence of alternative assembly signals. These findings support the belief that GABA(A) receptor assembly occurs via defined pathways to limit the receptor diversity.

The ability of differing subunit combinations of 5-HT3 receptors to form functional cell surface receptors was analyzed by a variety of approaches. The results revealed that 5-HT3 receptor assembly occurred within the endoplasmic reticulum (ER) and involved the interaction with chaperone proteins. The 5-HT3A subunit could assemble into functional homomeric receptors that were expressed on the cell surface. In contrast, the 5-HT3B subunit did not exhibit 5-hydroxytryptamine binding or function, could not assemble, and was efficiently retained and degraded within the ER. However, upon the coexpression of the 5-HT3A subunit, 5-HT3B could be “rescued” from the ER and transported to the cell surface to form functional heteromeric receptors with distinct functional characteristics. In support of the existence of homomeric 5-HT3 receptors in vivo, recombinantly expressed 5-HT3A receptors were capable of clustered cell surface expression in cortical neurons.

5-HT3 receptors are members of the Cys-loop superfamily of ligand-gated ion channels that include receptors for acetylcholine, GABA and glycine. Their native agonist binding sites exist at the interface between two subunits, within the extracellular domain of a pentameric ion channel. In all cases, the binding site is constructed from six distinct extracellular domains. Three regions (loops A–C) are on the primary face of one subunit and the remaining three regions (loops D–F) exist on the complementary face of the neighbouring subunit. Understanding the molecular structure of these receptors will facilitate the development of subunit-specific therapeutic agents. This is an important goal given that different receptor types control distinct biological functions. For example, GABAA receptor α subunits contribute to the distinct behavioural responses to benzodiazepines (binding at the α−γ interface), such as the anxiolytic (α2−γ2) and the sedative (α1−γ2) effects, while the α4 and α6 subunits do not contribute to benzodiazepine modulation. Agonist-binding sites generally exist at the interface of two distinct subunits in heteromeric receptors: α−β (GABAARs), α−γ or α−δ (nAChRs), α−β (glycine) and 3A–3B (5-HT3Rs). In the case of the 5-HT3Rs, this is inferred from the experimental results from the other receptor families. 5-HT3A/3B heteromeric receptors differ from homomeric 5-HT3A receptors in their single channel conductance, voltage dependence, desensitisation kinetics and permeability to Ca2+, but exhibit similar pharmacology. The similar pharmacology between 5-HT3A and 5-HT3A/3B receptors implies that the two subunits either have similar binding sites or that 5-HT3B does not contribute to 5-HT binding. Previous work on the stoichiometry of the 5-HT3A/3B receptor, using atomic force microscopy of epitoped-tagged recombinant subunits, concluded that the angle of bound antibodies did not support the existence of A–A interfaces but did support the existence of B–B interfaces (Barrera et al 2005). These results suggest that the most likely subunit order is B–B–A–B–A (a stoichiometry of (3B)3:(3A)2). However, in this issue of The Journal of Physiology, Thompson, Price & Lummis (2011), provide evidence that the 3B subunit does not contribute to the ligand-binding site. This conclusion is based on the failure of 3B subunit mutations at key sites in loops A–F to influence ligand binding, while the homologous mutations in 3A affected binding in both homomeric 5-HT3A and heteromeric 5-HT3A3B receptors. It is not clear what the explanation is for the contradiction to the previous study (Barrera et al 2005), but it may reflect the production of mixed stoichiometries of receptors. In this study (Thompson et al. 2011), only functional receptor (assembled and surface-expressed) are considered. In contrast, Barrera et al. (2005) included endoplasmic reticulum (ER) retained receptors. The receptor conformation proposed in their study lacks an A–A interface (B–B–A–B–A) and based on the findings of Thompson et al. (2011) they would be expected to be non-functional. However, it remains possible that 3B may use atypical residues within loops A–F to contribute to agonist binding. The results of Thompson et al. (2011) do not permit a full clarification of the heteromeric receptor order or stoichiometry. Nevertheless, as all other Cys-loop receptors require the binding of at least two agonist molecules, the most likely candidate (if only A–A interfaces can produce binding sites) is B–B–A*A*A (asterisks denoting agonist binding sites) and a stoichiometry of (3A)3:(3B)2). However, given that the B subunit can contribute to spontaneous activity, a second agonist-binding site may not be necessary. In which case, B–B–B–A*A and/or A*A–B–A–B could also exist. Thus, at present, we cannot answer the question; both 2Bs and 3Bs are possible. Regardless of whether the 5-HT3B contributes to 5-HT binding, it clearly impacts its coupling to channel gating. Moreover, it may confer altered receptor trafficking, localisation and association with accessory proteins, as observed for the γ2 subunit of GABAARs, which does not contribute to GABA binding. The lack of the 5-HT3B contribution to ligand binding may have consequences for the expression of 5-HT3 receptors. We proposed (Connolly & Wafford 2004) that receptor biogenesis must have the fidelity of receptor function at its heart. Consistent with this, the location of assembly signals overlaps the regions of ligand-binding sites; a cytoplasmic ER retention signal within 5-HT3B overlaps the ion selectivity filter; and the cytoplasmic ion pore, which is generated at cytoplasmic subunit interfaces, exists within a critical structural element that is a prerequisite for receptor biogenesis. Does this hypothesis have a practical consequence? Intriguingly, the chronic exposure to nicotine, GABA and 5-HT appears to promote the assembly of their respective receptors (Lester et al 2009; Eshaq et al 2010). Such a mechanism is attractive as it provides the opportunity to test the fidelity of receptors before their release from the ER. It may also influence the selection of particular receptor combinations based on agonist affinity (Lester et al 2009). This could be especially important in cells expressing multiple subunits, creating a specific supply on demand, and manipulating this process may offer novel therapeutic opportunities, based on subunit-selective compounds. If the 5-HT3B does not contribute to agonist binding, perhaps a pharmacological chaperone effect of 5-HT might operate preferentially on 5-HT3A homomers at the expense of 5-HT3B (or 5-HT3C-E) and so alter physiological responses. Such a consequence may be important in situations of high 5-HT levels, such as the therapeutic use of selective serotonin reuptake inhibitors and in gastrointestinal disorders.

The expression of alpha and beta gamma-aminobutyric acid type A receptor subunits produces GABA-gated channels which require the incorporation of either the gamma2 or gamma3 subunit for benzodiazepine modulation. Here we examine the role of the gamma2 subunit splice variants, gamma2S and gamma2L which differ by eight amino acids in the major intracellular domain, in mediating cell surface expression. Using immunocytochemistry we have demonstrated that when expressed alone, the gamma2S subunit can access the cell surface and internalize constitutively. In contrast, alpha1, beta2 and gamma2L are retained predominantly in the endoplasmic reticulum (ER) when expressed alone. Replacing the insert which differentiates gamma2L from gamma2S (LLRMFSFK) with eight alanines produces a phenotype identical to gamma2S. Both gamma2 subunits fail to produce high molecular weight oligomers observed for alpha1beta2 and alpha1beta2gamma2 heterooligomers and do not form functional ion channels. Surface expression of gamma2S is repressed upon the coexpression of alpha1 or beta2 subunits, resulting in ER-retained heterooligomers, suggesting that homomeric gamma2S is unlikely to occur in vivo. However, its independent maturation to surface competence and preferential assembly with alpha and beta subunits may ensure the production of functional benzodiazepine-sensitive receptors. Furthermore, the presence of the gamma2 subunit appears to confer an endocytotic capacity to these heterooligomeric receptors.

Activation of cAMP-dependent protein kinase (PKA) can enhance or reduce the function of neuronal GABAA receptors, the major sites of fast synaptic inhibition in the brain. This differential regulation depends on PKA-induced phosphorylation of adjacent conserved sites in the receptor beta subunits. Phosphorylation of beta 3 subunit-containing receptors at S408 and S409 enhanced the GABA-activated response, whereas selectively mutating S408 to alanine converted the potentiation into an inhibition, comparable to that of beta 1 subunits, which are phosphorylated solely on S409. These distinct modes of regulation were interconvertible between beta 1 and beta 3 subunits and depended upon the presence of S408 in either subunit. In contrast, beta 2 subunit-containing receptors were not phosphorylated or affected by PKA. Differential regulation by PKA of postsynaptic GABAA receptors containing different beta subunits may have profound effects on neuronal excitability.

1. Modulation of GABA(A) receptors by external H(+) was examined in cultured rat sympathetic neurones, and in Xenopus laevis oocytes and human embryonic kidney (HEK) cells expressing recombinant GABA(A) receptors composed of combinations of alpha 1, beta 1, beta 2, gamma 2S and delta subunits. 2. Changing the external pH from 7.4 reduced GABA-activated currents in sympathetic neurones. pH titration of the GABA-induced current was fitted with a pH model which predicted that H(+) interact with two sites (PK(a) values of 6.4 and 7.2). 3. For alpha 1 beta 1 GABA(A) receptors, low external pH (< 7.4) enhanced responses to GABA. pH titration predicted the existence of two sites with PK(a) values of 6.6 and 7.5. The GABA concentration-response curve was shifted to the left by low pH and non-competitively inhibited at high pH (> 7.4). 4. alpha 1 beta 1 gamma 2S receptor constructs were not affected by external pH, whereas exchanging the beta 1 subunit for beta 2 conferred a sensitivity to pH, with predicted PK(a) values of 5.16 and 9.44. 5. Low pH enhanced the responses to GABA on alpha 1 beta 1 delta subunits, whilst high pH caused an inhibition (PK(a) values of 6.6 and 9.9). The GABA concentration-response curves were enhanced (pH 5.4) or reduced (pH 9.4) with no changes in the GABA EC(50). 6. Immunoprecipitation with subunit and epitope-specific antisera to alpha 1, beta 1 and delta subunits demonstrated that these subunits could co-assemble in cell membranes. 7. Expression of alpha 1 beta 1 gamma 2S delta constructs resulted in a 'bell-shaped' pH titration relationship. Increasing or decreasing external pH inhibited the responses to GABA. 8. The pH sensitivity of recombinant GABA(A) receptors expressed in HEK cells was generally in accordance with data accrued from Xenopus oocytes. However, rapid application of GABA to alpha 1 beta 1 constructs at high pH (> 7.4) caused an increased peak and reduced steady-state current, with a correspondingly increased rate of desensitization. 9. Modulation of GABA(A) receptor function was apparently unaffected by the internal pH. Moreover, pH values between 5 and 9.5 did not significantly affect the charge distribution on the zwitterionic GABA molecules. 10. In conclusion, this study demonstrates that external pH can either enhance, have little effect, or reduce GABA-activated responses, and this is apparently dependent on the receptor subunit composition. The potential importance of H(+) sensitivity of GABA(A) receptors is discussed.

It is well established that a proportion of newly synthesized lysosomal enzymes and class II major histocompatibility complex antigens are delivered directly to the endocytic pathway from the Golgi complex. Here we show that a significant proportion of newly synthesized transferrin receptors can be detected in endosomes before reaching the cell surface. These newly synthesized transferrin receptors are delivered to the endosome more efficiently than either constitutively secreted soluble proteins or glycophosphatidylinositol-anchored plasma membrane proteins suggesting that their transfer to the endosome is signal-dependent. Identification of a signal-dependent transfer step for proteins like the transferrin receptor operating on the exocytic pathway has important implications for membrane biogenesis, especially in the establishment of cell surface polarity.