Professor Isabel Bermudez-Diaz

Nicotinic acetylcholine receptors (nAChRs) are a family of ligand-gated ion channels found in many organisms contributing to cognition, memory, and motor control. The pharmacological targeting of these receptors, using small molecules or peptides, presents an important strategy for the development of drugs that can treat important human diseases including neurodegenerative disorders. The Aplysia californica acetylcholine binding protein (Ac-AChBP) is a structural surrogate of the nAChR with high homology to the extracellular ligand binding domain of homopentameric nAChRs. In this study, we optimized protein painting-based mass spectrometry to identify regions of interaction between the Ac-AChBP and several nAChR ligands. Using molecular dyes that adhere to the surface of a solubilized Ac-AChBP complex, we identified amino acid residues that constitute a contact site within the Ac-AChBP for α-bungarotoxin, choline, nicotine, and amyloid-beta 1–42. By integrating innovation in protein painting mass spectrometry with computational structural modeling we present a new experimental tool for analyzing protein interactions of the nAChR.

NS9283, 3-(3-pyridyl)-5-(3-cyanophenyl)-1,2,4-oxadiazole, is a selective positive allosteric modulator of (α4)3(β2)2 nicotinic acetylcholine receptors (nAChR). It has good subtype selective therapeutic potential afforded by its specific binding to the unique α4-α4 subunit interface present in 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 2 the (α4)3(β2)2 nAChR. However, there is currently a lack of structure activity relationships (SAR) studies aimed at developing a class of congeners endowed with the same profile of activity that can help consolidate the druggability of the α4-α4 subunit interface. In this study, new NS9283 analogues were designed, synthesized, and characterized for their ability to selectively potentiate the ACh activity at heterologous (α4)3(β2)2 nAChRs vs nAChR subtypes (α4)2(β2)3, α5α4β2 and α7. With few exceptions, all the NS9283 analogues exerted positive modulation of the (α4)3(β2)2 nAChR AChevoked responses. Above all, those modified at the 3-cyanophenyl moiety by replacement with 3nitrophenyl (4), 4-cyanophenyl (10), and N-formyl-4-piperidinyl (20) showed the same efficacy as NS9283, although with lower potency. Molecular dynamic simulations of NS9283 and some selected analogues highlighted consistency between potentiation activity and pose of the ligand inside the α4α4 site with the main interaction being with the complementary (-) side and induction of a significant conformational change of the Trp156 residue in the principal (+) side.  

Recent work putatively linked a rare genetic variant of the chaperone Resistant to Inhibitors of acetylcholinesterase (RIC3) (NM_024557.4:c.262G>A, NP_078833.3:p.G88R) to a unique ability to speak backwards, a language skill that is associated with exceptional working memory capacity. RIC3 is important for the folding, maturation and functional expression of α7 nicotinic acetylcholine receptors (nAChR). We compared and contrasted the effects of RIC3G88R on assembly, cell surface expression and function of human α7 receptors using fluorescent protein tagged α7 nAChR and Förster resonance energy transfer (FRET) microscopy imaging in combination with functional assays and 125I-α-bungarotoxin binding. As expected, the wildtype RIC3 protein was found to increase both cell surface and functional expression of α7 receptors. In contrast, the variant form of RIC3 decreased both. FRET analysis showed that RICG88R increased the interactions between RIC3 and α7 protein in the Endoplasmic Reticulum. These results provide interesting and novel data to show that a RIC3 variant alters the interaction of RIC3 and α7, which translates to decreased cell surface and functional expression of α7 nAChR.

The quinuclidine scaffold has been extensively used for development of nicotinic acetylcholine receptor (nAChR) agonists, with hydrophobic substituents at position 3 of the quinuclidine framework providing selectivity for α7 nAChRs. In this study, six new ligands (4-9) containing a 3-(pyridin-3-yloxy)quinuclidine moiety (ether quinuclidine) were synthesised to gain a better understanding of the structural-functional properties of ether quinuclidines. To evaluate the pharmacological activity of these ligands, two-electrode voltage-clamp and single-channel recordings were performed. Only ligand 4 activated α7 nAChR. Ligand 5 and 7 had no effects on α7 nAChR but ligands 6, 8 and 9 potentiated the currents evoked by ACh. Ligand 6, was the most potent and efficacious of the potentiating ligands, with an estimated EC50 for potentiation of 12.6 ± 3.32 µM and a maximal potentiation of EC20 ACh responses of 850 ± 120 %.  Ligand 6 increased the maximal ACh responses without changing the kinetics of the current responses. At the single-channel level, the potentiation exerted by ligand 6 was evidenced at the low micromolar concentration range by the appearance of prolonged bursts of channel openings. Furthermore, computational studies revealed the preference of ligand 6 for an inter-subunit site in the transmembrane domain and highlighted some putative key interactions that explain the different profiles of the synthesised ligands. Notably, Met276 in the 15’ position of the transmembrane domain two almost abolished the effects of ligand 6 when mutated to Leu. We conclude that ligand 6 is a novel type I positive allosteric modulator (PAM) of a7 nAChR.

The α7 nicotinic acetylcholine receptor (nAChR) is present in neuronal and non-neuronal cells and has anti-inflammatory actions. Molecular dynamics simulations suggested that α7 nAChR interacts with a region of the SARS-CoV-2 spike protein (S), and a potential contribution of nAChRs to COVID-19 pathophysiology has been proposed. We applied whole-cell and single-channel recordings to determine whether a peptide corresponding to the Y674-R685 region of the S protein can directly affect α7 nAChR function. The S fragment exerts a dual effect on α7. It activates α7 nAChRs in the presence of positive allosteric modulators, in line with our previous molecular dynamics simulations showing favourable binding of this accessible region of the S protein to the nAChR agonist binding site. The S fragment also exerts a negative modulation of α7, which is evidenced by a profound concentration-dependent decrease in the durations of openings and activation episodes of potentiated channels and in the amplitude of macroscopic responses elicited by ACh. Our study identifies a potential functional interaction between α7 nAChR and a region of the S protein, thus providing molecular foundations for further exploring the involvement of nAChRs in COVID-19 pathophysiology.

The negative health effects cigarette smoking during pregnancy (SDP) on the foetus are well known. Despite previous reports of poor cognitive performance in offspring exposed to SDP, few studies specifically consider language outcomes according to maternal smoking. In this study, we systematically review the literature to assess the relationships between SDP and child language. Of the 14 studies reviewed, 13 (93%) reported significant associations between maternal smoking or exposure and language outcomes. Despite this consistent association, only 8 of the 13 studies reporting associations (62%) concluded direct relationships between exposure and outcome. The remaining studies suggested that the relationship between smoking and language could be explained by factors such as maternal IQ, socioeconomic status (SES) and parental age. Future studies should apply careful study designs allowing for confounding factors across child, parental, environmental and genetic influences. Our review suggests that smoking cessation is likely to positively affect child language outcomes.

Insect nicotinic acetylcholine receptors (nAChRs) are molecular targets of highly effective insecticides such as neonicotinoids. Functional expression of these receptors provides useful insights into their functional and pharmacological properties. Here, we report that the a5 nAChR subunit of the honey bee, Apis mellifera, functionally expresses in Xenopus laevis oocytes, which is the first time a homomeric insect nAChR has been robustly expressed in a heterologous system without the need for chaperone proteins. Using two-electrode voltage-clamp electrophysiology we show that the a5 receptor has low sensitivity to acetylcholine with an EC50 of 2.37 mM. However, serotonin acts as an agonist with a considerably lower EC50 at 119 microM that is also more efficacious than acetylcholine in activating the receptor. Molecular modelling indicates that residues in the complementary binding site may be involved in the selectivity towards serotonin. This is the first report of a ligand-gated ion channel activated by serotonin from an insect and phylogenetic analysis shows that the a5 subunit of A. mellifera and other non-Dipteran insects, including pest species, belong to a distinct subgroup of subunits, which may represent targets for the development of novel classes of insecticides.

Changeux et al. recently suggested that the SARS-CoV-2 spike protein may interact withn nicotinic acetylcholine receptors (nAChRs), and that such interactions may be involved in pathology and
infectivity. This hypothesis is based on the fact that the SARS-CoV-2 spike protein contains a sequence motif similar to known nAChR antagonists. Here, we use molecular simulations of validated atomically detailed structures of nAChRs, and of the spike, to investigate the possible binding of the Y674-R685 region of the spike to nAChRs. We examine the binding of the Y674-R685 loop to three nAChRs, namely the human α4β2 and α7 subtypes and the muscle-like αβγδ receptor from Tetronarce californica. Our results predict that Y674-R685 has affinity for nAChRs. The region of the spike responsible for binding contains a PRRA motif, a four-residue insertion not found in other SARS-like coronaviruses. The conformational behaviour of the bound Y674-R685 is highly dependent on the receptor subtype: it adopts extended conformations in the α4β2 and α7 complexes, but is more compact when bound to the muscle-like receptor. In the α4β2 and αβγδ complexes, the interaction of Y674-R685 with the receptors forces the loop C region to adopt an open conformation, similar to other known nAChR antagonists. In contrast, in the α7 complex, Y674-R685 penetrates deeply into the binding pocket where it forms interactions with the residues lining the aromatic box, namely with TrpB, TyrC1 and TyrC2. Estimates of binding energy suggest that Y674-R685 forms stable
complexes with all three nAChR subtypes. Analyses of simulations of the glycosylated spike show that the Y674-R685 region is accessible for binding. We suggest a potential binding orientation of the spike protein with nAChRs, in which they are in a non-parallel arrangement to one another. 

Background and purpose. The  α7 and α4ß2* (* denotes possibly assembly with another subunit) nicotinic acetylcholine receptors (nAChR) are the most abundant nAChR in the mammalian brain. These receptors are also the most commonly targeted nAChR in drug discovery programs for brain disorders. However, the development of subtype-specific agonists remains challenging, mainly due to the high degree of sequence homology and conservation of function in nAChR. We have developed C(10) variants of cytisine, a partial agonist of α4ß2 nAChR that has been used for smoking cessation. The C(10) methyl analogue used in this study displays negligible affinity for α7 nAChR, while retaining high-affinity for α4ß2 nAChR.

Experimental approach. The structural underpinning of the selectivity of 10-methylcytisine for α7 and α4ß2 nAChR was investigated using molecular dynamics simulations, mutagenesis and whole-cell and single-channel current recordings.

Key results. We identified a conserved arginine in the ß3-strand that exhibits a non-conserved function in nAChR. In α4ß2 nAChR, the arginine forms a salt-bridge with an aspartate residue in loop B that is necessary for receptor expression, whereas in α7 nAChR, this residue is not stabilised by electrostatic interactions, making its side chain highly mobile. This lack of constrain produces steric clashes with agonists and affects the dynamics of residues involved in agonist binding and the coupling network.

Conclusions and implications. We conclude that the high mobility of the ß3-strand arginine in the α7 nAChR influences agonist binding, and possibly gating network and desensitisation. The findings have implications for rational design of subtype-selective nAChR agents. 

Nicotinic acetylcholine receptors (nAChRs) modulate synaptic activity in the central nervous system. The α7 subtype, in particular, has attracted considerable interest in drug discovery as a target for several conditions, including Alzheimer’s disease and schizophrenia. Identifying agonist-induced structural changes underlying nAChR activation is fundamentally important for understanding biological function and rational drug design. Here, extensive equilibrium and nonequilibrium molecular dynamics simulations, enabled by cloud-based high-performance computing, reveal the molecular mechanism by which structural changes induced by agonist unbinding are transmitted within the human α7 nAChR. The simulations reveal the sequence of coupled structural changes involved in driving conformational change responsible for biological function. Comparison with simulations of the α4β2 nAChR subtype identifies features of the dynamical architecture common to both receptors, suggesting a general structural mechanism for signal propagation in this important family of receptors.

Brain nicotinic acetylcholine receptors (nAChRs), a heterogeneous family of pentameric acetylcholine-gated cation channels, have been suggested as molecular targets for the treatment of alcohol abuse and dependence. Here, we examined the effect of the competitive nAChR antagonist UFR2709 on the alcohol consumption of high-alcohol-drinking UChB rats. UChB rats were given free access to ethanol for 24-h periods in a two-bottle free-choice paradigm and their ethanol and water intake were measured. The animals were i.p injected daily for 17 days with a 10, 5, 2.5 or 1 mg/kg dose of UFR2709. Potential confounding motor effects of UFR2709 were assessed by examining the locomotor activity of animals administered the highest dose of UR2709 tested (10 mg/kg i.p.). UFR2709 reduced ethanol consumption and ethanol preference and increased water consumption in a dose-dependent manner. The most effective dose of UFR2709 was 2.5 mg/kg, which induced a 56% reduction in alcohol consumption. Administration of UFR2709 did not affect the weight or locomotor activity of the rats, suggesting that its effects on alcohol consumption and preference were mediated by specific nAChRs.

The use of positive allosteric modulators (PAM) of α7 nicotinic receptors is a promising therapy for neurodegenerative, inflammatory and cognitive disorders. Flavonoids are polyphenolic compounds showing neuroprotective, anti-inflammatory and pro-cognitive actions. Besides their well-known antioxidant activity, flavonoids trigger intracellular pathways and interact with receptors, including α7. To reveal how the beneficial actions of flavonoids are linked to α7 function, we evaluated the effects of three representative flavonoids -genistein, quercetin and the neoflavonoid 5,7-dihydroxy-4-phenylcoumarin- on whole-cell and single-channel currents. All flavonoids increase the maximal currents elicited by acetylcholine with minimal effects on desensitization and do not reactivate desensitized receptors, a behaviour consistent with type I PAMs. At the single-channel level, they increase the duration of the open state and produce activation in long-duration episodes with a rank order of efficacy of genistein > quercetin ≥ neoflavonoid. By using mutant and chimeric α7 receptors, we demonstrated that flavonoids share transmembrane structural determinants with other PAMs. The α7-PAM activity of flavonoids results in decreased cell levels of reactive oxygen species. Thus, allosteric potentiation of α7 may be an additional mechanism underlying neuroprotective actions of flavonoids, which may be used as scaffolds for designing new therapeutic agents.

Neuronal nicotinic receptors containing α4 and β2 subunits assemble in two pentameric stoichiometries, (α4)3(β2)2 and (α4)2(β2)3, each with distinct pharmacological signatures; (α4)3(β2)2 receptors are strongly potentiated by the drug NS9283, whereas (α4)2(β2)3 receptors are unaffected. Despite this stoichiometry-selective pharmacology, the molecular identity of the target for NS9283 remains elusive. Here, studying (α4)3(β2)2 receptors, we show that mutations at either the principal face of the β2 subunit or the complementary face of the α4 subunit prevent NS9283 potentiation of ACh-elicited single-channel currents, suggesting the drug targets the β2–α4 pseudo-agonist sites, the α4–α4 agonist site, or both sites. To distinguish among these possibilities, we generated concatemeric receptors with mutations at specified subunit interfaces, and monitored the ability of NS9283 to potentiate ACh-elicited single-channel currents. We find that a mutation at the principal face of the β2 subunit at either β2–α4 pseudo-agonist site suppresses potentiation, whereas mutation at the complementary face of the α4 subunit at the α4–α4 agonist site allows a significant potentiation. Thus, monitoring potentiation of single concatemeric receptor channels reveals that the β2–α4 pseudo-agonist sites are required for stoichiometry-selective drug action. Together with the recently determined structure of the (α4)3(β2)2 receptor, the findings have implications for structure-guided drug design.

Neuronal nicotinic receptors containing α4 and β2 subunits assemble in two pentameric stoichiometries, (α4)3(β2)2 and (α4)2(β2)3, each with distinct pharmacological signatures; (α4)3(β2)2 receptors are strongly potentiated by the drug NS9283, whereas (α4)2(β2)3 receptors are unaffected. Despite this stoichiometry-selective pharmacology, the molecular identity of the target for NS9283 remains elusive. Here, studying (α4)3(β2)2 receptors, we show that mutations at either the principal face of the β2 subunit or the complementary face of the α4 subunit prevent NS9283 potentiation of ACh-elicited single-channel currents, suggesting the drug targets the β2–α4 pseudo-agonist sites, the α4–α4 agonist site, or both sites. To distinguish among these possibilities, we generated concatemeric receptors with mutations at specified subunit interfaces, and monitored the ability of NS9283 to potentiate ACh-elicited single-channel currents. We find that a mutation at the principal face of the β2 subunit at either β2–α4 pseudo-agonist site suppresses potentiation, whereas mutation at the complementary face of the α4 subunit at the α4–α4 agonist site allows a significant potentiation. Thus, monitoring potentiation of single concatemeric receptor channels reveals that the β2–α4 pseudo-agonist sites are required for stoichiometry-selective drug action. Together with the recently determined structure of the (α4)3(β2)2 receptor, the findings have implications for structure-guided drug design.

The fifth subunit in the (α4β2)2α4 nicotinic acetylcholine receptor (nAChR) plays a determining role in the pharmacology of this nAChR type. Here, we have examined the role of the fifth subunit in the ACh responses of the (α4β2)2β2 nAChR type.

EXPERIMENTAL APPROACH:

The role of the fifth subunit in receptor function was explored using two-electrode voltage-clamp electrophysiology, along with subunit-targeted mutagenesis and the substituted cysteine scanning method applied to fully linked (α4β2)2β2 receptors.

KEY RESULTS:

Covalent modification of cysteine substituted fifth subunit with a thiol-reactive agent (MTS) caused irreversible inhibition of receptor function. ACh reduced the rate of MTS reaction but the competitive inhibitor dihydro-β-erythroidine had no effect. Alanine substitution of conserved residues that line the core of agonist sites on α4(+)/β2(-) interfaces did not impair receptor function. However, impairment of agonist binding to α4(+)/β2(-) agonist sites by mutagenesis modified the effect of ACh on the rate of MTS reaction. The extent of this effect was dependent on the position of the agonist site relative to the fifth subunit.

CONCLUSIONS AND IMPLICATIONS:

We conclude that the fifth subunit in (α4β2)2β2 receptor isoform modulates maximal ACh responses. This effect appears to be driven by a modulatory, and asymmetric, association with the α4(+)/β2(-) agonist sites.

The nAChR belongs to the Cys loop family of ligand gated ion channels. It assembles as a pentameric structure of one of two stoichiometries, (a4b2)2a4 and (a4b2)2b2. These receptors respond to ACh with low and high sensitivity, respectively. The ACh binding sites are located at the a4(þ)/b2(-) interfaces in the N-terminal region of the protein, which is extracellularly positioned. The (a4b2)2a4 receptor has been recently shown to house an additional functional

ACh binding site at its signature a4(þ)/a4(þ) interface. The remaining interfaces, b2(þ)/a4(-) interfaces, are thought to bind allosteric modulators, but their contribution to receptor function is not known. However, it has been speculated, on the basis of the role of equivalent interfaces in allostery in other Cys loop ligand gated ion channels such as the GABAA receptor and a3b2 nAChR) that they may house binding sites for allosteric modulators.

Using fully concatenated (a4b2)2a4 nACh receptors in conjunction with functional mutagenesis and substituted cystine accesibility methods (SCAM) we have examined whether the b2(þ)/a4(-) interfaces impact receptor function. Our results suggest that b2(þ)/a4(-) interface in the (a4b2)2a4 nAChR receptors may be a central part of an allosteric pathway that modulates the function of the a4(þ)/a4(-) interface.

Neuronal nicotinic acetylcholine receptors (nAChRs) containing the α5 subunit modulate nicotine consumption, and the human CHRNA5 rs16969968 polymorphism, causing the replacement of the aspartic acid residue at position 398 with an asparagine (α5DN), has recently been associated with increased use of tobacco and higher incidence of lung cancer. We show that in ventral midbrain neurons, the α5 subunit is essential for heteromeric nAChR-induced intracellular-free Ca(2+) concentration elevations and that in α5(-/-) mice, a class of large-amplitude nicotine-evoked currents is lost. Furthermore, the expression of the α5DN subunit is not able to restore nicotinic responses, indicating a loss of function by this subunit in native neurons. To understand how α5DN impairs heteromeric nAChR functions, we coexpressed α4, α5, or α5DN subunits with a dimeric concatemer (β2α4) in a heterologous system, to obtain nAChRs with fixed stoichiometry. Both α5(β2α4)2 and α5DN(β2α4)2 nAChRs yielded similar levels of functional expression and Ca(2+) permeability, measured as fractional Ca(2+) currents (8.2 ± 0.7% and 8.0 ± 1.9%, respectively), 2-fold higher than α4(β2α4)2. Our results indicate that the loss of function of nicotinic responses observed in α5DN-expressing ventral midbrain neurons is neither due to an intrinsic inability of this subunit to form functional nAChRs nor to an altered Ca(2+) permeability but likely to intracellular modulation.

A mutation in the second transmembrane domain of the GABA receptor subunit, Rdl, is associated with resistance to insecticides such as dieldrin and fipronil. Molecular cloning of Rdl cDNA from a strain of the malaria mosquito, Anopheles gambiae, which is highly resistant to dieldrin revealed this mutation (A296G) as well as another mutation in the third transmembrane domain (T345M). Wild-type, A296G, T345M and A296G + T345M homomultimeric Rdl were expressed in Xenopus laevis oocytes and their sensitivities to fipronil, deltamethrin, 1,1,1-trichloro-2,2-bis(p-chlorophenyl) ethane (DDT), imidacloprid and spinosad were measured using two-electrode voltage-clamp electrophysiology. Spinosad and DDT had no agonist or antagonist actions on Rdl. However, fipronil, deltamethrin and imidacloprid decreased GABA-evoked currents. These antagonistic actions were either reduced or abolished with the A296G and the A296G + T345M mutations while T345M alone appeared to have no significant effect. In conclusion, this study identifies another mutation in the mosquito Rdl that is associated with insecticide resistance. While T345M itself does not affect insecticide sensitivity, it may serve to offset the structural impact of A296G. The present study also highlights Rdl as a potential secondary target for neonicotinoids and pyrethroids.

For decades, the development of selective compounds has been the main goal for chemists and biologists involved in drug discovery. However, diverse lines of evidence indicate that polypharmacological agents, i.e. those that act simultaneously at various protein targets, might show better profiles than selective ligands, regarding both efficacy and side effects. On the other hand, the availability of the crystal structure of different receptors allows a detailed analysis of the main interactions between drugs and receptors in a specific binding site. Neuronal nicotinic acetylcholine receptors (nAChRs) constitute a large and diverse family of ligand-gated ion channels (LGICs) that, as a product of its modulation, regulate neurotransmitter release, which in turns produce a global neuromodulation of the central nervous system. nAChRs are pentameric protein complexes in such a way that expression of compatible subunits can lead to various receptor assemblies or subtypes. The agonist binding site, located at the extracellular region, exhibits different properties depending on the subunits that conform the receptor. In the last years, it has been recognized that nAChRs could also contain one or more allosteric sites which could bind non-classical nicotinic ligands including several therapeutically useful drugs. The presence of multiple binding sites in nAChRs offers an interesting possibility for the development of novel polypharmacological agents with a wide spectrum of actions.

The α4β2 nicotinic acetylcholine receptor (nAChR) is the most abundant nAChR type in the brain, and this receptor type exists in alternate (α4β2)2α4 and (α4β2)2β2 forms, which are activated by agonists with strikingly differing efficacies. Recent breakthroughs have identified an additional operational agonist binding site in the (α4β2)2α4 nAChR that is responsible for the signature sensitivity of this receptor to activation by agonists, yet the structural mechanisms determining agonist efficacy at this receptor type are not yet fully understood. In this study, we characterized the ligand selectivity of the individual agonist sites of the (α4β2)2α4 nAChR to determine whether differences in agonist selectivity influence agonist efficacy. Applying the substituted cysteine accessibility method to individual agonist sites in concatenated (α4β2)2α4 receptors, we determined the agonist selectivity of the agonist sites of the (α4β2)2α4 receptor. We show that (a) accessibility of substituted cysteines to covalent modification by methanesulfonate reagent depends on the agonist site at which the modification occurs and (b) that agonists such as sazetidine-A and TC-2559 are excluded from the site at the α4/α4 interface. Given that additional binding to the agonist site in the α4/α4 interface increases acetylcholine efficacy and that agonists excluded from the agonist site at the α4/α4 interface behave as partial agonists, we conclude that the ability to engage all agonist sites in (α4β2)2α4 nAChRs is a key determinant of agonist efficacy. The findings add another level of complexity to the structural mechanisms that govern agonist efficacy in heteromeric nAChRs and related ligand-gated ion channels.

Heteropentameric neuronal nicotinic receptors assemble so that the canonical acetylcholine-binding sites are located at the interfaces between two pairs of subunits, while the fifth subunit does not participate in a canonical transmitter-binding site. Several subunits are considered to be unable to participate in forming a functional receptor when they occupy a position that would contribute to such a site, including the α5 subunit. The α5 subunit is of interest because of its apparent involvement in nicotine dependence and in the control of dopamine release. We have examined this question using α4 and β2 subunits in concatemeric constructs with the α5 subunit, expressed in Xenopus oocytes. Using dimeric constructs of α4 and β2 subunits expressed with free α5 and pentameric constructs incorporating a single copy of α5, we find that the α5 subunit can occupy the position of a nonbinding subunit, or replace a β2 subunit participating in a canonical binding site. The resulting receptors functionally resemble pentamers assembled with two copies of α4 and three copies of β2. Functional receptors apparently cannot be formed with α5 subunits in both canonical binding sites. These observations extend the present ideas on the possible positions in the pentamer that may be occupied by the α5 subunit, and suggest that additional physiologic or pharmacological subtypes of neuronal nicotinic receptors may be present in neurons.

Nicotine directly regulates striatal dopamine (DA) neurotransmission via presynaptic nicotinic acetylcholine receptors (nAChRs) that are α6β2 and/or α4β2 subunit-containing, depending on region. Chronic nicotine exposure in smokers upregulates striatal nAChR density, with some reports suggesting differential impact on α6- or α4-containing nAChRs. Here, we explored whether chronic nicotine exposure modifies striatal DA transmission, whether the effects of acute nicotine on DA release probability persist and whether there are modifications to the regulation of DA release by α6-subunit-containing (*) relative to non-α6* nAChRs in nucleus accumbens (NAc) and in caudate-putamen (CPu). We detected electrically evoked DA release at carbon-fiber microelectrodes in striatal slices from mice exposed for 4-8 weeks to nicotine (200 μg/mL in saccharin-sweetened drinking water) or a control saccharin solution. Chronic nicotine exposure subtly reduced striatal DA release evoked by single electrical pulses, and in NAc enhanced the range of DA release evoked by different frequencies. Effects of acute nicotine (500 nm) on DA release probability and its sensitivity to activity were apparent. However, in NAc there was downregulation of the functional dominance of α6-nAChRs (α6α4β2β3), and an emergence in function of non-α6* nAChRs. In CPu, there was no change in the control of DA release by its α6 nAChRs (α6β2β3) relative to non-α6. These data suggest that chronic nicotine subtly modifies the regulation of DA transmission, which, in NAc, is through downregulation of function of a susceptible population of α6α4β2β3 nAChRs. This imbalance in function of α6:non-α6 nAChRs might contribute to DA dysregulation in nicotine addiction.

Nicotine is an agonist of nicotinic acetylcholine receptors (nAChRs) that has been extensively used as a template for the synthesis of α4β2-preferring nAChRs. Here, we used the N-methyl-pyrrolidine moiety of nicotine to design and synthesise novel α4β2-preferring neonicotinic ligands. We increased the distance between the basic nitrogen and aromatic group of nicotine by introducing an ester functionality that also mimics acetylcholine (Fig. 2). Additionally, we introduced a benzyloxy group linked to the benzoyl moiety. Although the neonicotinic compounds fully inhibited binding of both [α-(125)I]bungarotoxin to human α7 nAChRs and [(3)H]cytisine to human α4β2 nAChRs, they were markedly more potent at displacing radioligand binding to human α4β2 nAChRs than to α7 nAChRs. Functional assays showed that the neonicotinic compounds behave as antagonists at α4β2 and α4β2α5 nAChRs. Substitutions on the aromatic ring of the compounds produced compounds that displayed marked selectivity for α4β2 or α4β2α5 nAChRs. Docking of the compounds on homology models of the agonist binding site at the α4/β2 subunit interfaces of α4β2 nAChRs suggested the compounds inhibit function of this nAChR type by binding the agonist binding site.

BACKGROUND AND PURPOSE:

Nicotinic ACh (α4β2)2α4 receptors are highly prone to desensitization by prolonged exposure to low concentrations of agonist. Here, we report on the sensitivity of the three agonist sites of the (α4β2)2α4 to desensitization induced by prolonged exposure to ACh. We present electrophysiological data that show that the agonist sites of the (α4β2)2α4 receptor have different sensitivity to desensitization and that full receptor occupation decreases sensitivity to desensitization.

EXPERIMENTAL APPROACH:

Two-electrode voltage-clamp electrophysiology was used to study the desensitization of concatenated (α4β2)2α4 receptors expressed heterologously in Xenopus oocytes. Desensitization was assessed by measuring the degree of functional inhibition caused by prolonged exposure to ACh, as measured under equilibrium conditions. We used the single-point mutation α4W182A to measure the contribution of individual agonist sites to desensitization.

KEY RESULTS:

(α4β2)2α4 receptors are less sensitive to activation and desensitization by ACh than (α4β2)2β2 receptors. Incorporation of α4W182A into any of the agonist sites of concatenated (α4β2)2α4 receptors decreased sensitivity to activation and desensitization but the effects were more pronounced when the mutation was introduced into the α4(+)/α4(-) interface.

CONCLUSIONS AND IMPLICATIONS:

The findings suggest that the agonist sites in (α4β2)2α4 receptors are not functionally equivalent. The agonist site at the α4(+)/α4(-) interface defines the sensitivity of (α4β2)2α4 receptors to agonist-induced activation and desensitization. Functional differences between (α4β2)2α4 and (α4β2)2β2 receptors might shape the physiological and behavioural responses to nicotinic ligands when the receptors are exposed to nicotinic ligands for prolonged periods of times.

Nicotinic acetylcholine receptor (nAChR) alpha 4 and beta 2 subunits assemble in two alternate stoichiometries to produce (alpha 4 beta 2)(2)alpha 4 and (alpha 4 beta 2)(2)alpha 4, which display different agonist sensitivities. Functionally relevant agonist binding sites are thought to be located at alpha 4(+)/beta 2(-) subunit interfaces, but because these interfaces are present in both receptor isoforms, it is unlikely that they account for differences in agonist sensitivities. In contrast, incorporation of either alpha 4 or beta 2 as auxiliary subunits produces isoform-specific alpha 4(+)/alpha 4(-) or beta 2(+)/beta 2(-) interfaces. Using fully concatenated (alpha 4 beta 2)(2)alpha 4 nAChRs in conjunction with structural modeling, chimeric receptors, and functional mutagenesis, we have identified an additional site at the alpha 4(+)/alpha 4(-) interface that accounts for isoform-specific agonist sensitivity of the (alpha 4 beta 2)(2)alpha 4 nAChR. The additional site resides in a region that also contains a potentiating Zn2+ site but is engaged by agonists to contribute to receptor activation. By engineering alpha 4 subunits to provide a free cysteine in loop C at the alpha 4(+)alpha 4(-) interface, we demonstrated that the acetylcholine responses of the mutated receptors are attenuated or enhanced, respectively, following treatment with the sulfhydryl reagent [2-(trimethylammonium)ethyl] methanethiosulfonate or aminoethyl methanethiosulfonate. The findings suggest that agonist occupation of the site at the alpha 4(+)/(alpha 4(-) interface leads to channel gating through a coupling mechanism involving loop C. Overall, we propose that the additional agonist site at the alpha 4(+)/alpha 4(-) interface, when occupied by agonist, contributes to receptor activation and that this additional contribution underlies the agonist sensitivity signature of (alpha 4 beta 2)(2)alpha 4 nAChRs.

Objective: To study the effect of dietary tryptophan (TRP) depletion

(DTD) on plasma hormones and brain 5-HT2 receptors in

the male rat and monitor resulting adaptive responses that might

occur.

Background: St John's wort is an effective antidepressant that can reduce tobacco withdrawal symptoms, but it is not known whether it assists cessation. Chromium assists weight loss and might limit post cessation weight gain. Methods: In a factorial design, we randomised smokers stopping smoking to 900 mg St John's wort (SJW) active or placebo and also randomised them to 400 mu m chromium or placebo daily. Treatment started 2 weeks prior to quit day and continued for 14 weeks. Participants and researchers were blind to treatment allocation. All participants received weekly behavioural Support. The primary endpoints were biochemically confirmed prolonged abstinence and mean weight gain in abstinent smokers 4 weeks after quitting. Results: 6/71 (8.5%) participants on active SJW and 9/72 (12.5%) on placebo achieved prolonged abstinence at 4 weeks, an odds ratio (OR) (95% confidence interval) of 0.65 (0.22-1.92). At 6 months, 3 (4.2%) SJW active and 6 (8.3%) SJW placebo participants were still abstinent, an OR of 0.49 (0.12-2.02). Among these participants, the mean difference in weight gain between active chromium and placebo was -0.81 kg (-3.79 to 2.18) at 4 weeks and -3.88 kg (-12.13 to 4.38) at 6 months. Conclusions: Taking together the absolute quit rates, the small difference between active and placebo, and lack of effects on withdrawal shows that SJW is ineffective for smoking cessation. Insufficient people stopped smoking to property test the efficacy of chromium in preventing weight gain, but the point estimate indicates a potentially worthwhile benefit.

(S)-Aporphine metho salts with the 1,2,9,10 oxygenation pattern displaced radioligands from recombinant human α7 and α4β2 neuronal nicotinic acetylcholine receptors (nAChR) at low micromolar concentrations. The affinity of the nonphenolic glaucine methiodide (4) (vs [3H]cytisine) was the lowest at α4β2 nAChR (Ki = 10 μM), and predicentrine methiodide (2) and xanthoplanine iodide (3), with free hydroxyl groups at C-2 or C-9, respectively, had the highest affinity at these receptors (Ki ≈ 1 μM), while the affinity of the diphenolic boldine methiodide (1) was intermediate between these values. At homomeric α7 nAChR, xanthoplanine had the highest affinity (Ki = 10 μM) vs [125I]α-bungarotoxin while the other three compounds displaced the radioligand with Ki values between 15 and 21 μM. At 100 μM, all four compounds inhibited the responses of these receptors to EC50 concentrations of ACh. The effects of xanthoplanine iodide (3) were studied in more detail. Xanthoplanine fully inhibited the EC50 ACh responses of both α7 and α4β2 nACh receptors with estimated IC50 values of 9 ± 3 μM (α7) and 5 ± 0.8 μM (α4β2).

Neuronal nicotinic acetylcholine receptors subserve predominantly modulatory roles in the brain, making them attractive therapeutic targets. Natural products provide key leads in the quest for nicotinic receptor subtype-selective compounds. Cytisine, found in Leguminosae spp., binds with high affinity to α4β2⁎ nicotinic receptors. We have compared the effect of C3 and C5 halogenation of cytisine and methylcytisine (MCy) on their interaction with native rat nicotinic receptors. 3-Bromocytisine (3-BrCy) and 3-iodocytisine (3-ICy) exhibited increased binding affinity (especially at α7 nicotinic receptors; Ki∼0.1 μM) and functional potency, whereas C5-halogenation was detrimental. 3-BrCy and 3-ICy were more potent than cytisine at evoking [3H]dopamine release from striatal slices (EC50 ∼11 nM), [3H]noradrenaline release from hippocampal slices (EC50 ∼250 nM), increases in intracellular Ca2+ in PC12 cells and inward currents in Xenopus oocytes expressing human α3β4 nicotinic receptor (EC50 ∼2 μM). These compounds were also more efficacious than cytisine. C3-halogenation of cytisine is proposed to stabilize the open conformation of the nicotinic receptor but does not enhance subtype selectivity.

S-Nicotine, the principal psychoactive constituent of Nicotiana tabacum, underpins addiction to tobacco smoking. Although tobacco consumption is a leading cause of death worldwide, nicotine itself is also proposed to have potential therapeutic benefits for a diverse range of conditions. Nicotine interacts with its cognate receptors in the central nervous system to exert a predominantly modulatory influence, making neuronal nicotinic receptors attractive therapeutic targets. Here, we focus on three natural products as lead compounds for drug discovery programs, nicotine, epibatidine and cytisine, and consider the aims and limitations that shape these drug discovery endeavors.

α4 and β2 nicotinic acetylcholine receptor (nAChR) subunits expressed heterologously assemble into receptors with high (HS) and low (LS) sensitivity to acetylcholine (ACh); their relative proportions depend on the α4to β2 ratio. In this study, injection of oocytes with 1:10 α4/β2 subunit cDNA ratios favored expression of HS α4β2 nAChRs, as evidenced by monophasic ACh concentration-response curves, whereas injections with 10:1 cDNA ratios favored expression of LS α4β2 receptors. The stoichiometry was inferred from the shifts in the ACh EC50 values caused by Leu to Thr mutations at position 9′ of the second transmembrane domain of α4 and β2. The 1:10 injection ratio produced the (α4)2(β2)3 stoichiometry, whereas 10:1 injections produced the (α4)3(β2)2 stoichiometry. The agonists epibatidine, 3-[2(S)-azetidinylmethoxy]pyridine (A-85380), 5-ethoxy-metanicotine (TC-2559), cytisine, and 3-Br-cytisine and the antagonists dihydro-β-erythroidine and d-tubocurarine were more potent at HS receptors. TC-2559 was more efficacious than ACh at HS receptors but was a partial agonist at LS receptors. Epibatidine was more efficacious than ACh at LS receptors and a partial agonist at HS receptors. Cytisine and 5-halogenated cytisines had moderate efficacy at LS receptors but had almost no efficacy at HS receptors. By exploiting the differential effects of ACh, TC-2559 and 5-I-cytisine we evaluated the effects of long-term exposure to nicotine on HS and LS receptors expressed in Xenopus laevis oocytes after cDNA injections or microtransplantation of α4β2 receptors assembled in human embryonic kidney 293 cells. We conclude that nicotine up-regulates HS α4β2 receptors, probably by influencing the assembly of receptors rather than by altering the functional state of LS α4β2 nAChRs.

Effects of cholinergic drugs on human α4β2 nicotinic acetylcholine receptors expressed in Xenopusoocytes have been investigated in electrophysiological and ligand binding experiments. Atropine, scopolamine, physostigmine, and tacrine combine potentiation of ion current induced by low concentrations of acetylcholine with inhibition of ion current evoked by high concentrations of acetylcholine. Rivastigmine, galanthamine, and dichlorvos cause only inhibition of ion current evoked by low concentrations of acetylcholine. Binding experiments show that the potentiating cholinergic drugs atropine, scopolamine, and physostigmine are competitive ligands of human α4β2 nicotinic acetylcholine receptors. Conversely, the inhibitory cholinergic drugs galanthamine and rivastigmine are non-competitive. The non-competitive drugs are not allosteric, since they do not affect the saturation curve of the radioligand [3H]cytisine. Effects of potentiating cholinergic drugs on nicotinic acetylcholine receptors are consistent with and predicted by a model comprising competitive drug effects at two equivalent agonist recognition sites on the nicotinic acetylcholine receptor combined with non-competitive ion channel block.

Effects of derivatives of coclaurine (C), which mimic the ‘eastern’ or the nonquaternary halves of the alkaloids tetrandrine or d-tubocurarine, respectively, both of which are inhibitors of nicotinic acetylcholine receptors (nACh), were examined on recombinant, human α7, α4β2 and α4β4 nACh receptors expressed in Xenopus oocytes and clonal cell lines using two-electrode voltage clamping and radioligand binding techniques.

In this limited series, Cs have higher affinity and are most potent at α4 subunit-containing-nACh receptors and least potent at homomeric α7 receptors, and this trend is very marked for the N-unsubstituted C and its O,O′-bisbenzyl derivative.

7-O-Benzyl-N-methylcoclaurine (BBCM) and its 12-O-methyl derivative showed the highest affinities and potencies at all three receptor subtypes, and this suggests that lipophilicity at C7 and/or C12 increases potency.

Laudanosine and armepavine (A) were noncompetitive and voltage-dependent inhibitors of α7, α4β2 or α4β4 receptors, but the bulkier C7-benzylated 7BNMC (7-O-benzyl-N-methylcoclaurine) and 7B12MNMC (7-O-benzyl-N,12-O-dimethyl coclaurine) were voltage-independent, noncompetitive inhibitors of nACh receptors. Voltage-dependence was also lost on going from A to its N-ethyl analogue.

These studies suggest that C derivatives may be useful tools for studies characterising the antagonist and ion channel sites on human α7, α4β2 or α4β4 nACh receptors and for revealing structure–function relationships for nACh receptor antagonists.

Rasmussen encephalitis (RE) sera were screened for antibodies to human α7 nicotinic acetylcholine receptors (nAChRs) using electrophysiology, calcium imaging, and ligand binding assays. Sera from two of nine patients with RE blocked ACh-induced currents through α7 nAChRs and the ACh-induced rise in intracellular free calcium ([Ca2+]i) and inhibited 125I-α-bungarotoxin binding in cells expressing α7 nAChRs. Thus, the α7 nAChR is a potential target for pathogenic antibodies in patients with RE

To determine the prevalence of serum antibodies to the ionotropic glutamate receptor 3 (GluR3) in patients with Rasmussen encephalitis (RE), a severe epileptic disorder, and to compare with serum from control subjects and patients with intractable epilepsy (IE).

METHODS:

The authors looked for serum immunoglobulin (Ig) G antibodies to GluR3 in 30 patients with RE, including two patients who had plasma exchange and 12 who had been treated with IV Igs with varying results, and 49 patients with IE and 23 healthy individuals, using ELISA with GluR3B peptide, Western blot analysis of recombinant full-length GluR3, immunoprecipitation of [35S]- and [125I]-labeled GluR3 extracellular domains, immunohistochemistry on rat brain sections, and electrophysiology of GluR3 expressed in Xenopus oocytes.

RESULTS:

Low levels of antibodies to the GluR3B peptide were detected using ELISA in only 4 of the 79 patients with epilepsy (2 with RE and 2 with IE); binding to GluR3B in other sera was shown to be nonspecific. One other patient with IE had antibodies to recombinant GluR3 on Western blot analysis. However, none of the sera tested precipitated either the [35S]- or the [125I]-labeled GluR3 domains; none bound to rat brain sections in a manner similar to rabbit antibodies to GluR3; and none of the nine sera tested affected the electrophysiologic function of GluR3.

CONCLUSIONS:

GluR3 antibodies were only infrequently found in Rasmussen encephalitis or intractable epilepsy.

The α7 nicotinic acetylcholine receptor (nAChR) plays a key role in neural development and neurodegeneration. Here, we identify a novel, modulatory receptor ligand, a 14-amino acid peptide (AEFHRWSSYMVHWK) derived from the C-terminus of acetylcholinesterase (AChE). In three different in vitro preparations, this ‘AChE-peptide’ is bioactive in a ligand-specific and concentration-dependent manner. First, it modulates acutely the effect of acetylcholine (ACh) on Xenopus oocytes transfected with human α7, but not α4/β2, nAChR. The action persists when intracellular calcium is chelated with BAPTA or when calcium is substituted with barium ions. This observation suggests that intracellular Ca2+ signals do not mediate the interaction between the peptide and nAChR, but rather that the interaction is direct: however, the intervention of other mediators cannot be excluded. Secondly, in recordings from the CA1 region in guinea-pig hippocampal slices, AChE-peptide modulates synaptic plasticity in a α-bungarotoxin (α-BgTx)-sensitive manner. Thirdly, in organotypic cultures of rat hippocampus, long-term exposure to peptide attenuates neurite outgrowth: this chronic, functional effect is selectively blocked by the α7 nAChR antagonists, α-BgTx and methyllycaconitine, but not by the α4/β2-preferring blocker dihydro-β-ethroidine. A scrambled peptide variant, and the analogous peptide from butyrylcholinesterase, are ineffective in all three paradigms. The consequences of this novel modulation of the α7 nAChR may be activation of a trophic-toxic axis, of relevance to neurodegeneration.

Human α7 nicotinic acetylcholine (ACh) receptors were expressed in Xenopus oocytes and the effects of the N-methyl-d-aspartate (NMDA) receptor open channel blockers memantine and cerestat on this receptor were examined using two-electrode voltage-clamp recordings and 125I-α-bungarotoxin (125I-α-bgtx) binding.

Memantine and cerestat produced complete inhibition of ACh-induced inward currents with affinities similar to that reported for native NMDA receptors. Cerestat, IC50 1.7 (−1; +2) μm, was more potent than memantine, IC50 5 (−3;+8) μm, and the effects of both drugs were fully and rapidly reversible.

Inhibition of α7 receptor function was voltage-independent, and it occurred at concentrations far lower than those needed to inhibit (never completely) binding of 125I-α-bgtx to α7 receptors, suggesting that the effects of memantine or cerestat are noncompetitive.

These results provide evidence that human α7 receptors are inhibited by memantine and cerestat and suggest that caution should be applied when using these compounds to study systems in which NMDA and nACh receptors co-exist.

Functional effects of the well-characterized antagonist of L-type Ca2+ channels tetrandrine on recombinant human γ-aminobutyric acid type A (GABAA) (α1β2γ2s) receptor or human α7, α4β2, α1β1δγ and α1β1δε nicotinic acetylcholine receptors expressed in Xenopus oocytes were examined using two-electrode voltage clamp. Tetrandrine inhibited the function of acetylcholine nicotinic receptors, but it had no effect on GABAA receptors. Potency of inhibition was influenced by the receptor subtype and the rank order was α4β2>α7>α1β1δγ≅α1β1δε. Functional inhibition of α4β2 and α1β1δγ receptors was noncompetitive, but only inhibition of α1β1δγ receptors was voltage-dependent. Binding of 125I-α-bungarotoxin to α1β1δγ or3H-cytisine to α4β2 receptors was also inhibited by tetrandrine, but inhibition was noncompetitive and required concentrations higher than those needed to inhibit receptor function. Inhibition of both α7 receptor function and binding of 125I-α-bungarotoxin to α7 receptor were mixed competitive/noncompetitive and occurred at a similar concentration range.

Effects of cytisine (cy), 3-bromocytisine (3-Br-cy), 5-bromocytisine (5-Br-cy) and 3,5-dibromocytisine (3,5-diBr-cy) on human (h) alpha7-, alpha4beta2- and alpha4beta4 nicotinic acetylcholine (nACh) receptors, expressed in Xenopus oocytes and cell lines, have been investigated. Cy and its bromo-isosteres fully inhibited binding of both [alpha-(125)I]bungarotoxin ([alpha-(125)I]BgTx) to halpha7- and [(3)H]cy to halpha4beta2- or halpha4beta4-nACh receptors. 3-Br-cy was the most potent inhibitor of both [alpha-(125)I]BgTx and [(3)H]cy binding. Cy was less potent than 3-Br-cy, but 5-Br-cy and 3,5-diBr-cy were the least potent inhibitors. Cy and 3-Br-cy were potent full agonists at halpha7-nACh receptors but behaved as partial agonists at halpha4beta2- and halpha4beta4-nACh receptors. 5-Br-cy and 3,5-diBr-cy had low potency and were partial agonists at halpha7- and halpha4beta4-nACh receptors, but they elicited no responses on halpha4beta2-nACh receptors. Cy and 3-Br-cy produced dual dose-response curves (DRC) at both halpha4beta2- and halpha4beta4-nACh receptors, but ACh produced dual DRC only at halpha4beta2-nACh receptors. Low concentrations of cy, 3-Br-cy and 5-Br-cy enhanced ACh responses of oocytes expressing halpha4beta2-nACh receptors, but at high concentrations they inhibited the responses. In contrast, 3,5-diBr-cy only inhibited, in a competitive manner, ACh responses of halpha4beta2-nACh receptors. It is concluded that bromination of the pyridone ring of cy produces marked changes in effects of cy that are manifest as nACh receptor subtype-specific differences in binding affinities and in functional potencies and efficacies.

Effects of the L-type calcium channel antagonist diltiazem on recombinant human GABA(A) receptor (alpha1beta2gamma2s) or on muscle (alpha1beta1deltagamma and alpha1beta1delta(epsilon)) or neuronal (alpha7 and alpha4beta2) nicotinic acetylcholine receptors expressed in Xenopus oocytes were examined using two-electrode voltage-clamp. Diltiazem inhibited the function of both muscle and neuronal nicotinic receptors, but it had no effect on GABA(A) receptors. The extent of functional inhibition of nicotinic receptors depended on the receptor subtype, and the order of inhibition potency by diltiazem was alpha7>alpha4beta2 approximately alpha1beta1deltagamma approximately alpha1beta1delta(epsilon). Inhibition of alpha7 receptor function was non-competitive and voltage-independent, and it occurred at concentrations far lower than those needed to inhibit (never completely) binding of (125)I-alpha-bungarotoxin to heterologously expressed alpha7 receptors in mammalian cells. Pre-incubation in diltiazem before concomitant application with acetylcholine increased inhibition of function and slowed recovery from inhibition. Verapamil, a phenylalkylamine antagonist of L-type Ca(2+) channels also fully inhibited alpha7 receptor function and partially inhibited (125)I-alpha-bungarotoxin binding to alpha7 receptors, but was less potent than diltiazem. Effects on both alpha7 receptor function and (125)I-alpha-bungarotoxin binding by verapamil plus diltiazem suggest separate sites for verapamil and diltiazem on alpha7 receptors. These results provide further evidence that L-type Ca(2+) channel drugs inhibit ligand-gated cationic channels and suggest that caution should be applied when using these compounds to study systems in which L-type Ca(2+) channels and ligand-gated cationic channels co-exist.

The mouse serotonin (5-HT) receptor subtype, 5-HT7, belongs to the family of seven transmembrane G-protein-coupled receptors. To identify the structural basis for the coupling of 5-HT7 receptor to GαS we constructed a number of receptor mutants in which amino acid residues were either substituted or deleted from the second and third intracellular loops. Wild-type and mutant 5-HT7 receptors were expressed in insect cells using the baculovirus vectors. Two mutant receptor species, 5-HT7(E325G) and 5-HT7(K327S), demonstrated markedly impaired abilities to stimulate adenylyl cyclase. The results suggest the importance of the C-terminal region of the third intracellular loop in receptor–G-protein interaction and that specific charged residues, E325 and K327, may play a critical role in this interaction.

The effects of gamma-hexachlorocyclohexane (gamma-HCH) and its alpha, beta and delta isomers on the gamma-aminobutyric acid (GABA) responses of human alpha1beta3gamma2S and alpha6beta3gamma2S GABA(A) receptors expressed in Xenopus oocytes were examined by conventional two-electrode voltage-clamp techniques. Gamma-HCH induced partial inhibition of EC50 GABA responses, whereas the alpha and delta isomers produced potentiation of EC20 GABA currents. In contrast, beta-HCH had no effect on GABA currents, even at concentrations as high as 100 microM. The effects of the active HCH isomers were not influenced by alpha subunit composition because there was no significant difference in either the inhibition or potentiation of alpha1beta3gamma2S or alpha6beta3gamma2S GABA(A) receptors. Delta- and gamma-HCH antagonized picrotoxin inhibition and caused displacement of specific [35S]t-butylbicyclophosphorothionate binding. Delta-HCH potentiation was found to be additive with steroid, loreclezole and lanthanum potentiation, but nonadditive with potentiation by pentobarbital and propofol, which suggested that its activity was linked to the barbiturate site.

Insect cells are routinely used for the production of receptor proteins. Expression of the Drosophila 5-HTdro1 serotonin receptor resulted in positive coupling of the receptor to adenylyl cyclase via the G(alpha)s G-protein subtype. The Drosophila 5-HTdro2B receptor stimulated the metabolism of inositol phospholipid via a pertussis toxin-insensitive G-protein, but exhibited no detectable inhibition of adenylyl cyclase. Immunoblot analysis of the endogenous G-proteins revealed that Sf9 cells lack the G-protein subtypes G(alpha i 1-3) and G(alpha)o, but express the subtype G(alpha)s and G(alpha)q.