Karen A. Bollan

The epilepsy mutation, γ2(R43Q) disrupts a highly conserved inter-subunit contact site, perturbing the biogenesis of GABAA receptors

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.

Chronic exposure to neonicotinoids increases neuronal vulnerability to mitochondrial dysfunction in the bumblebee (Bombus terrestris)

The global decline in the abundance and diversity of insect pollinators could result from habitat loss, disease, and pesticide exposure. The contribution of the neonicotinoid insecticides (e.g., clothianidin and imidacloprid) to this decline is controversial, and key to understanding their risk is whether the astonishingly low levels found in the nectar and pollen of plants is sufficient to deliver neuroactive levels to their site of action: the bee brain. Here we show that bumblebees (Bombus terrestris andax) fed field levels [10 nM, 2.1 ppb (w/w)] of neonicotinoid accumulate between 4 and 10 nM in their brains within 3 days. Acute (minutes) exposure of cultured neurons to 10 nM clothianidin, but not imidacloprid, causes a nicotinic acetylcholine receptor‐dependent rapid mitochondrial depolarization. However, a chronic (2 days) exposure to 1 nM imidacloprid leads to a receptor‐dependent increased sensitivity to a normally innocuous level of acetylcholine, which now also causes rapid mitochondrial depolarization in neurons. Finally, colonies exposed to this level of imidacloprid show deficits in colony growth and nest condition compared with untreated colonies. These findings provide a mechanistic explanation for the poor navigation and foraging observed in neonicotinoid treated bumblebee colonies.—Moffat, C., Pacheco, J. G., Sharp, S., Samson, A. J., Bollan, K. A., Huang, J., Buckland, S. T., Connolly, C. N. Chronic exposure to neonicotinoids increases neuronal vulnerability to mitochondrial dysfunction in the bumblebee (Bombus terrestris). FASEB J. 29, 2112‐2119 (2015). www.fasebj.org

Differential Subcellular Localization of RIC-3 Isoforms and Their Role in Determining 5-HT3 Receptor Composition

RIC-3 has been identified as a chaperone molecule involved in promoting the functional expression of nicotinic acetylcholine and 5-HT3 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.

An Asymmetric Contribution to γ-Aminobutyric Type A Receptor Function of a Conserved Lysine within TM2–3 of α1, β2, and γ2 Subunits

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.

The promiscuous role of the epsilon subunit in GABAA receptor biogenesis.

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 (epsilon-beta2-alpha1-beta2-gamma2) receptors, or at position 4 (replaces beta2) in alphabetaepsilon (alpha1-beta2-alpha1-epsilon-beta2) receptors. In both cases, it appears that only a single GABA binding site is present.

An asymmetric contribution to GABAa receptor function of a conserved lysine within TM2-3 of α1, β2 and γ2 subunits

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.

Neonicotinoids target distinct nicotinic acetylcholine receptors and neurons, leading to differential risks to bumblebees

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.

5-HT Cellular Sequestration During Chronic Exposure Delays 5-HT3 Receptor Resensitization Due to its Subsequent Release.

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.

5-Hydroxytryptamine (5-HT) Cellular Sequestration during Chronic Exposure Delays 5-HT3 Receptor Resensitization due to Its Subsequent Release

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.