Neuroprotective amyloid ß N-terminal peptides differentially alter human α7- and α7ß2-nicotinic acetylcholine (nACh) receptor single-channel properties.

BACKGROUND AND PURPOSE: Oligomeric amyloid ß 1-42 (oAß1-42) exhibits agonist-like action at human α7- and α7ß2-containing nicotinic receptors. The N-terminal amyloid ß1-15 fragment (N-Aß fragment) modulates presynaptic calcium and enhances hippocampal-based synaptic plasticity via α7-containing nicotinic receptors. Further, the N-Aß fragment and its core sequence, the N-amyloid-beta core hexapeptide (N-Aßcore), protect against oAß1-42-associated synapto- and neurotoxicity. Here, we investigated how oAß1-42, the N-Aß fragment, and the N-Aßcore regulate the single-channel properties of α7- and α7ß2-nicotinic receptors. EXPERIMENTAL APPROACH: Single-channel recordings measured the impact of acetylcholine, oAß1-42, the N-Aß fragment, and the N-Aßcore on the unitary properties of human α7- and α7ß2-containing nicotinic receptors expressed in nicotinic-null SH-EP1 cells. Molecular dynamics simulations identified potential sites of interaction between the N-Aß fragment and orthosteric α7+/α7- and α7+/ß2- nicotinic receptor binding interfaces. KEY RESULTS: The N-Aß fragment and N-Aßcore induced α7- and α7ß2-nicotinic receptor single-channel openings. Relative to acetylcholine, oAß1-42 preferentially enhanced α7ß2-nicotinic receptor single-channel open probability and open-dwell times. Co-application with the N-Aßcore neutralized these effects. Further, administration of the N-Aß fragment alone, or in combination with acetylcholine or oAß1-42, selectively enhanced α7-nicotinic receptor open probability and open-dwell times (compared to acetylcholine or oAß1-42). CONCLUSIONS AND IMPLICATIONS: Amyloid-beta peptides demonstrate functional diversity in regulating α7- and α7ß2-nicotinic receptor function, with implications for a wide range of nicotinic receptor-mediated functions in Alzheimer's disease. The effects of these peptides on α7- and/or α7ß2-nicotinic receptors revealed complex interactions with these subtypes, providing novel insights into the neuroprotective actions of amyloid ß-derived fragments against the toxic effects of oAß1-42.

Levo-tetrahydropalmatine inhibits α4ß2 nicotinic receptor response to nicotine in cultured SH-EP1 cells.

Nicotine, a major component of tobacco, is highly addictive and acts on nicotinic acetylcholine receptors (nAChRs) to stimulate reward-associated circuits in the brain. It is well known that nAChRs play critical roles in mediating nicotine reward and addiction. Current FDA-approved medications for smoking cessation are the antidepressant bupropion and the nicotinic partial agonist varenicline, yet both are limited by adverse side effects and moderate efficacy. Thus, development of more efficacious medications with fewer side effects for nicotine addiction and smoking cessation is urgently needed. l-Tetrahydropalmatine (l-THP) is an active ingredient of the Chinese medicinal herb Corydalis ambigua that possesses rich neuropharmacological actions on dopamine (DA) receptors in the mesocorticolimbic dopaminergic reward pathway. L-THP has been explored as anti-addiction treatments for drug abuse including nicotine. However, the targets and mechanisms of l-THP-caused anti-nicotine effects are largely unknown. In this study we address this question by elucidating the effects of l-THP on human neuronal nAChRs using patch-clamp recordings. Human neuronal α4ß2-nAChRs were heterologously expressed in SH-EP1 human epithelial cells. Bath application of nicotine (0.1-100 µM) induced inward currents, co-application of l-THP (3 µM) inhibited nicotine-induced currents in the transfected cells. L-THP-caused inhibition was concentration-dependent (the EC50 values for inhibiting the peak and steady-state current were 18 and 2.1 µM, respectively) and non-competitive. Kinetic analysis of the whole-cell currents showed that l-THP slowed rising time and accelerated decay time constants. L-THP specifically modulated α4ß2-nAChRs, as it did not affect α7-nAChRs or α1*-nAChRs (muscle type). Interestingly, two putative α4ß2-nAChR isoforms, namely sazetidine A-activated, high-sensitive one (α42ß23-nAChR) and cytisine-activated, low-sensitive one (α43ß22-nAChR) were pharmacologically separated, and the low-sensitive one was more susceptible to l-THP inhibition than the high-sensitive one. In conclusion, we demonstrate that l-THP blocks neuronal α4ß2-nAChR function, which may underlie its inhibition on nicotine addiction.

Sleep-related hypermotor epilepsy associated mutations uncover important kinetic roles of α4ß2- nicotinic acetylcholine receptor intracellular structures.

Sleep-related hypermotor epilepsy (SHE) is a group of seizure disorders prominently associated with mutations in nicotinic acetylcholine receptors (nAChR). The most prevalent central nervous system nAChR subtype contains α4 and ß2 subunits, in two ratios. (α4ß2)2ß2-nAChR have high agonist sensitivity (HS-isoform), whereas (α4ß2)2α4-nAChR agonist responses exhibit a small high-sensitivity, and a predominant low-sensitivity, phase of function (LS-isoform). Multiple non-synonymous mutations in the second and third transmembrane domains of α4 and ß2 subunits are associated with SHE. We recently demonstrated that two additional, SHE-associated, missense mutations in the major cytoplasmic loops of these subunits [α4(R336H) and ß2(V337G)] cause increased macroscopic function-per receptor. Here, we use single-channel patch-clamp electrophysiology to show that these mutations influence single-channel amplitudes and open- and closed-state kinetics. Pure populations of HS- or LS-isoform α4ß2-nAChR were expressed by injecting either 1:10 or 30:1 α4:ß2 cRNA ratios, respectively, into Xenopus laevis oocytes. Functional properties of the resulting mutant α4ß2-nAChR isoforms were compared to their wildtype counterparts. α4(R336H) subunit incorporation minimally affected single-channel amplitudes, whereas ß2(V337G) subunit incorporation reduced them significantly in both isoforms. However, for both mutant subunits, increased function-per-receptor was predominantly caused by altered single channel kinetics. The α4(R336H) mutation primarily destabilizes desensitized states between openings. By contrast, the ß2(V337G) mutation principally stabilizes receptor open states. The use of naturally-occurring and physiologically-impactful mutations has allowed us to define valuable new insights regarding the functional roles of nAChR intracellular domains. Further mechanistic context is provided by intracellular-domain structures recently published for other members of the Cys-loop receptor superfamily (α3ß4-nAChR and 5-HT3AR).

Implications of Oligomeric Amyloid-Beta (oAß42) Signaling through α7ß2-Nicotinic Acetylcholine Receptors (nAChRs) on Basal Forebrain Cholinergic Neuronal Intrinsic Excitability and Cognitive Decline.

Neuronal and network-level hyperexcitability is commonly associated with increased levels of amyloid-ß (Aß) and contribute to cognitive deficits associated with Alzheimer's disease (AD). However, the mechanistic complexity underlying the selective loss of basal forebrain cholinergic neurons (BFCNs), a well-recognized characteristic of AD, remains poorly understood. In this study, we tested the hypothesis that the oligomeric form of amyloid-ß (oAß42), interacting with α7-containing nicotinic acetylcholine receptor (nAChR) subtypes, leads to subnucleus-specific alterations in BFCN excitability and impaired cognition. We used single-channel electrophysiology to show that oAß42 activates both homomeric α7- and heteromeric α7ß2-nAChR subtypes while preferentially enhancing α7ß2-nAChR open-dwell times. Organotypic slice cultures were prepared from male and female ChAT-EGFP mice, and current-clamp recordings obtained from BFCNs chronically exposed to pathophysiologically relevant level of oAß42 showed enhanced neuronal intrinsic excitability and action potential firing rates. These resulted from a reduction in action potential afterhyperpolarization and alterations in the maximal rates of voltage change during spike depolarization and repolarization. These effects were observed in BFCNs from the medial septum diagonal band and horizontal diagonal band, but not the nucleus basalis. Last, aged male and female APP/PS1 transgenic mice, genetically null for the ß2 nAChR subunit gene, showed improved spatial reference memory compared with APP/PS1 aged-matched littermates. Combined, these data provide a molecular mechanism supporting a role for α7ß2-nAChR in mediating the effects of oAß42 on excitability of specific populations of cholinergic neurons and provide a framework for understanding the role of α7ß2-nAChR in oAß42-induced cognitive decline.

Cocaine potently blocks neuronal α3ß4 nicotinic acetylcholine receptors in SH-SY5Y cells.

Cocaine is one of the most abused illicit drugs worldwide. It is well known that the dopamine (DA) transporter is its major target; but cocaine also acts on other targets including nicotinic acetylcholine receptors (nAChRs). In this study, we investigated the effects of cocaine on a special subtype of neuronal nAChR, α3ß4-nAChR expressed in native SH-SY5Y cells. α3ß4-nAChR-mediated currents were recorded using whole-cell recordings. Drugs were applied using a computer-controlled U-tube drug perfusion system. We showed that bath application of nicotine induced inward currents in a concentration-dependent manner with an EC50 value of 20 µM. Pre-treatment with cocaine concentration-dependently inhibited nicotine-induced current with an IC50 of 1.5 µM. Kinetic analysis showed that cocaine accelerated α3ß4-nAChR desensitization, which caused a reduction of the amplitude of nicotine-induced currents. Co-application of nicotine and cocaine (1.5 µM) depressed the maximum response on the nicotine concentration-response curve without changing the EC50 value, suggesting a non-competitive mechanism. The cocaine-induced inhibition of nicotine response exhibited both voltage- and use-dependence, suggesting an open-channel blocking mechanism. Furthermore, intracellular application of GDP-ßS (via recording electrode) did not affect cocaine-induced inhibition, suggesting that cocaine did not alter receptor internalization. Moreover, intracellular application of cocaine (30 µM) failed to alter the nicotine response. Finally, cocaine (1.5 µM) was unable to inhibit the nicotine-induced inward current in heterologous expressed α6/α3ß2ß3-nAChRs and α4ß2-nAChRs expressed in human SH-EP1 cells. Collectively, our results suggest that cocaine is a potent blocker for native α3ß4-nAChRs expressed in SH-SY5Y cells.

Attenuation in Nicotinic Acetylcholine Receptor α9 and α10 Subunit Double Knock-Out Mice of Experimental Autoimmune Encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is attenuated in nicotinic acetylcholine receptor (nAChR) α9 subunit knock-out (α9 KO) mice. However, protection is incomplete, raising questions about roles for related, nAChR α10 subunits in ionotropic or recently-revealed metabotropic contributions to effects. Here, we demonstrate reduced EAE severity and delayed onset of disease signs in nAChR α9/α10 subunit double knock-out (DKO) animals relative to effects in wild-type (WT) control mice. These effects are indistinguishable from contemporaneously-observed effects in nicotine-treated WT or in α9 KO mice. Immune cell infiltration into the spinal cord and brain, reactive oxygen species levels in vivo, and demyelination, mostly in the spinal cord, are reduced in DKO mice. Disease severity is not altered relative to WT controls in mice harboring a gain-of-function mutation in α9 subunits. These findings minimize the likelihood that additional deletion of nAChR α10 subunits impacts disease differently than α9 KO alone, whether through ionotropic, metabotropic, or alternative mechanisms. Moreover, our results provide further evidence of disease-exacerbating roles for nAChR containing α9 subunits (α9*-nAChR) in EAE inflammatory and autoimmune responses. This supports our hypothesis that α9*-nAChR or their downstream mediators are attractive targets for attenuation of inflammation and autoimmunity.

Distinctive single-channel properties of α4ß2-nicotinic acetylcholine receptor isoforms.

Central nervous system nicotinic acetylcholine receptors (nAChR) are predominantly of the α4ß2 subtype. Two isoforms exist, with high or low agonist sensitivity (HS-(α4ß2)2ß2- and LS-(α4ß2)2α4-nAChR). Both isoforms exhibit similar macroscopic potency and efficacy values at low acetylcholine (ACh) concentrations, mediated by a common pair of high-affinity α4(+)/(-)ß2 subunit binding interfaces. However LS-(α4ß2)2α4-nAChR also respond to higher concentrations of ACh, acting at a third α4(+)/(-)α4 subunit interface. To probe isoform functional differences further, HS- and LS-α4ß2-nAChR were expressed in Xenopus laevis oocytes and single-channel responses were assessed using cell-attached patch-clamp. In the presence of a low ACh concentration, both isoforms produce low-bursting function. HS-(α4ß2)2ß2-nAChR exhibit a single conductance state, whereas LS-(α4ß2)2α4-nAChR display two distinctive conductance states. A higher ACh concentration did not preferentially recruit either conductance state, but did result in increased LS-(α4ß2)2α4-nAChR bursting and reduced closed times. Introduction of an α4(+)/(-)α4-interface loss-of-function α4W182A mutation abolished these changes, confirming this site's role in mediating LS-(α4ß2)2α4-nAChR responses. Small or large amplitude openings are highly-correlated within individual LS-(α4ß2)2α4-nAChR bursts, suggesting that they arise from distinct intermediate states, each of which is stabilized by α4(+)/(-)α4 site ACh binding. These findings are consistent with α4(+)/(-)α4 subunit interface occupation resulting in allosteric potentiation of agonist actions at α4(+)/(-)ß2 subunit interfaces, rather than independent induction of high conductance channel openings.

Alpha6-containing nicotinic acetylcholine receptor is a highly sensitive target of alcohol.

Alcohol use disorder (AUD) is a serious public health problem that results in tremendous social, legal and medical costs to society. Unlike other addictive drugs, there is no specific molecular target for ethanol (EtOH). Here, we report a novel molecular target that mediates EtOH effects at concentrations below those that cause legally-defined inebriation. Using patch-clamp recording of human α6*-nicotinic acetylcholine receptor (α6*-nAChR) function when heterologously expressed in SH-EP1 human epithelial cells, we found that 0.1-5 mM EtOH significantly enhances α6*-nAChR-mediated currents with effects that are dependent on both EtOH and nicotine concentrations. EtOH exposure increased both whole-cell current rising slope and decay constants. This EtOH modulation was selective for α6*-nAChRs since it did not affect α3ß4-, α4ß2-, or α7-nAChRs. In addition, 5 mM EtOH also increased the frequency and amplitude of dopaminergic neuron transients in mouse brain nucleus accumbens slices, that were blocked by the α6*-nAChR antagonist, α-conotoxin MII, suggesting a role for native α6*-nAChRs in low-dose EtOH effects. Collectively, our data suggest that α6*-nAChRs are sensitive targets mediating low-dose EtOH effects through a positive allosteric mechanism, which provides new insight into mechanisms involved in pharmacologically-relevant alcohol effects contributing to AUD.

Roles of Nicotine in the Development of Intracranial Aneurysm Rupture.

Background and Purpose- Tobacco cigarette smoking is considered to be a strong risk factor for intracranial aneurysmal rupture. Nicotine is a major biologically active constituent of tobacco products. Nicotine's interactions with vascular cell nicotinic acetylcholine receptors containing α7 subunits (α7*-nAChR) are thought to promote local inflammation and sustained angiogenesis. In this study, using a mouse intracranial aneurysm model, we assessed potential contributions of nicotine exposure and activation of α7*-nAChR to the development of aneurysmal rupture. Methods- Intracranial aneurysms were induced by a combination of deoxycorticosterone-salt induced hypertension and a single-dose elastase injection into cerebrospinal fluid in mice. Results- Exposure to nicotine or an α7*-nAChR-selective agonist significantly increased aneurysm rupture rate. Coexposure to an α7*-nAChR antagonist abolished nicotine's deleterious effect. In addition, nicotine's promotion of aneurysm rupture was absent in smooth muscle cell-specific α7*-nAChR subunit knockout mice but not in mice lacking α7*-nAChR on endothelial cells or macrophages. Nicotine treatment increased the mRNA levels of vascular endothelial growth factor, platelet-derived growth factor-B, and inflammatory cytokines. α7*-nAChR antagonist reversed nicotine-induced upregulation of these growth factors and cytokines. Conclusions- Our findings indicate that nicotine exposure promotes aneurysmal rupture through actions on vascular smooth muscle cell α7*-nAChR.

α1-FANGs: Protein Ligands Selective for the α-Bungarotoxin Site of the α1-Nicotinic Acetylcholine Receptor.

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels that play a central role in neuronal and neuromuscular signal transduction. Here, we have developed FANG ligands, fibronectin antibody-mimetic nicotinic acetylcholine receptor-generated ligands, using mRNA display. We generated a 1 trillion-member primary e10FnIII library to target a stabilized α1 nicotinic subunit (α211). This library yielded 270000 independent potential protein binding ligands. The lead sequence, α1-FANG1, represented 25% of all library sequences, showed the highest-affinity binding, and competed with α-bungarotoxin (α-Btx). To improve this clone, a new library based on α1-FANG1 was subjected to heat, protease, binding, off-rate selective pressures, and point mutations. This resulted in α1-FANG2 and α1-FANG3. These proteins bind α211 with KD values of 3.5 nM and 670 pM, respectively, compete with α-Btx, and show improved subunit specificity. α1-FANG3 is thermostable ( Tm = 62 °C) with a 6 kcal/mol improvement in folding free energy compared with that of the parent α1-FANG1. α1-FANG3 competes directly with the α-Btx binding site of intact neuromuscular heteropentamers [(α1)2ß1γδ] in mammalian culture-derived cellular membranes and in Xenopus laevis oocytes expressing these nAChRs. This work demonstrates that mRNA display against a monomeric ecto-domain of a pentamer has the capability to select ligands that bind that subunit in both a monomeric and a pentameric context. Overall, our work provides a route to creating a new family of stable, well-behaved proteins that specifically target this important receptor family.

Pharmacological and functional comparisons of α6/α3ß2ß3-nAChRs and α4ß2-nAChRs heterologously expressed in the human epithelial SH-EP1 cell line.

Neuronal nicotinic acetylcholine receptors containing α6 subunits (α6*-nAChRs) show highly restricted distribution in midbrain neurons associated with pleasure, reward, and mood control, suggesting an important impact of α6*-nAChRs in modulating mesolimbic functions. However, the function and pharmacology of α6*-nAChRs remain poorly understood because of the lack of selective agonists for α6*-nAChRs and the challenging heterologous expression of functional α6*-nAChRs in mammalian cell lines. In particular, the α6 subunit is commonly co-expressed with α4*-nAChRs in the midbrain, which masks α6*-nAChR (without α4) function and pharmacology. In this study, we systematically profiled the pharmacology and function of α6*-nAChRs and compared these properties with those of α4ß2 nAChRs expressed in the same cell line. Heterologously expressed human α6/α3 chimeric subunits (α6 N-terminal domain joined with α3 trans-membrane domains and intracellular loops) with ß2 and ß3 subunits in the human SH-EP1 cell line (α6*-nAChRs) were used. Patch-clamp whole-cell recordings were performed to measure these receptor-mediated currents. Functionally, the heterologously expressed α6*-nAChRs exhibited excellent function and showed distinct nicotine-induced current responses, such as kinetics, inward rectification and recovery from desensitization, compared with α4ß2-nAChRs. Pharmacologically, α6*-nAChR was highly sensitive to the α6 subunit-selective antagonist α-conotoxin MII but had lower sensitivity to mecamylamine and dihydro-ß-erythroidine. Nicotine and acetylcholine were found to be full agonists for α6*-nAChRs, whereas epibatidine and cytisine were determined to be partial agonists. Heterologously expressed α6*-nAChRs exhibited pharmacology and function distinct from those of α4ß2-nAChRs, suggesting that α6*-nAChRs may mediate different cholinergic signals. Our α6*-nAChR expression system can be used as an excellent cell model for future investigations of α6*-nAChR function and pharmacology.

Distinctive Roles for α7*- and α9*-Nicotinic Acetylcholine Receptors in Inflammatory and Autoimmune Responses in the Murine Experimental Autoimmune Encephalomyelitis Model of Multiple Sclerosis.

Previous studies have demonstrated immunosuppressive and anti-inflammatory effects of nicotine, including in the experimental autoimmune encephalomyelitis (EAE) model in mice of some forms of multiple sclerosis (MS). Other studies using knock-out (KO) mice have implicated nicotinic acetylcholine (ACh) receptors containing α7, α9, or ß2 subunits (α7*-, α9*- or ß2*-nAChR) in different, disease-exacerbating or disease-ameliorating processes. These outcomes are in harmony with gene expression analyses showing nAChR subunit mRNA in many classes of immune system cell types. Consistent with influences on disease status, predictable effects of nAChR subunit (and subtype) KO, or of nicotine exposure, are seen on immune cell numbers and distribution and on cytokine levels or other markers of immunity, inflammation, demyelination, and axonal degradation. Providing support for our hypotheses about distinctive roles for nAChR subtypes in EAE, here we have used direct and adoptive EAE induction and a nAChR subunit gene double knock-out (DKO) strategy. Immune cell expression of nAChR α9 subunits as protein is demonstrated by immunostaining of isolated CD4+, CD8+, CD11b+ and CD11c+ cells from wild-type (WT) mice, but not in cells from nAChR α9 subunit KO animals. Nicotine exposure is protective against directly-induced EAE in WT or α7/α9 DKO animals relative to effects seen in WT/vehicle-treated mice, but, remarkably, EAE is exacerbated in vehicle-treated α7/α9 DKO mice. Brain lesion volume and intra-cranial inflammatory activity similarly are higher in DKO/vehicle than in WT/vehicle-treated animals, although nicotine's protective effects are seen in each instance. By contrast, in adoptive transfer studies, disease severity is attenuated and disease onset is delayed in recipients of splenocytes from WT animals treated with nicotine rather than with vehicle. Moreover, protection as seen in nicotine-treated WT animals is the same in recipients of splenocytes from nAChR α7/α9 DKO mice irrespective of their exposure to nicotine or vehicle. When combined with previous observations, these findings are consistent with disease exacerbation (or even induction) being mediated at least in part via α9*-nAChR in peripheral immune cells. They also suggest protective roles of central nervous system (CNS) α7*-nAChR. The results suggest that both α7*- and α9*-nAChR are potential targets of therapeutic ligands to modulate inflammation and autoimmunity.

Isoform-specific mechanisms of α3ß4*-nicotinic acetylcholine receptor modulation by the prototoxin lynx1.

This study investigates-for the first time to our knowledge-the existence and mechanisms of functional interactions between the endogenous mammalian prototoxin, lynx1, and α3- and ß4-subunit-containing human nicotinic acetylcholine receptors (α3ß4*-nAChRs). Concatenated gene constructs were used to express precisely defined α3ß4*-nAChR isoforms (α3ß4)2ß4-, (α3ß4)2α3-, (α3ß4)2α5(398D)-, and (α3ß4)2α5(398N)-nAChR in Xenopus oocytes. In the presence or absence of lynx1, α3ß4*-nAChR agonist responses were recorded by using 2-electrode voltage clamp and single-channel electrophysiology, whereas radioimmunolabeling measured cell-surface expression. Lynx1 reduced (α3ß4)2ß4-nAChR function principally by lowering cell-surface expression, whereas single-channel effects were primarily responsible for reducing (α3ß4)2α3-nAChR function [decreased unitary conductance (≥50%), altered burst proportions (3-fold reduction in the proportion of long bursts), and enhanced closed dwell times (3- to 6-fold increase)]. Alterations in both cell-surface expression and single-channel properties accounted for the reduction in (α3ß4)2α5-nAChR function that was mediated by lynx1. No effects were observed when α3ß4*-nAChRs were coexpressed with mutated lynx1 (control). Lynx1 is expressed in the habenulopeduncular tract, where α3ß4*-α5*-nAChR subtypes are critical contributors to the balance between nicotine aversion and reward. This gives our findings a high likelihood of physiologic significance. The exquisite isoform selectivity of lynx1 interactions provides new insights into the mechanisms and allosteric sites [α(-)-interface containing] by which prototoxins can modulate nAChR function.-George, A. A., Bloy, A., Miwa, J. M., Lindstrom, J. M., Lukas, R. J., Whiteaker, P. Isoform-specific mechanisms of α3ß4*-nicotinic acetylcholine receptor modulation by the prototoxin lynx1.

Synthesis and biological evaluation of novel hybrids of highly potent and selective α4ß2-Nicotinic acetylcholine receptor (nAChR) partial agonists.

We previously reported the cyclopropylpyridine and isoxazolylpyridine ether scaffolds to be versatile building blocks for creating potent α4ß2 nicotinic acetylcholine receptor (nAChR) partial agonists with excellent selectivity over the α3ß4 subtype. In our continued efforts to develop therapeutic nicotinic ligands, seven novel hybrid compounds were rationally designed, synthesized, and evaluated in [3H]epibatidine binding competition studies. Incorporation of a cyclopropane- or isoxazole-containing side chain onto the 5-position of 1-(pyridin-3-yl)-1,4-diazepane or 2-(pyridin-3-yl)-2,5-diazabicyclo[2.2.1]heptane led to highly potent and selective α4ß2* nAChR partial agonists with Ki values of 0.5-51.4 nM for α4ß2 and negligible affinities for α3ß4 and α7. Moreover, compounds 21, 25, and 30 maintained the functional profiles (EC50 and IC50 values of 15-50 nM) of the parent azetidine-containing compounds 3 and 4 in the 86Rb+ ion flux assays. In vivo efficacy of the most promising compound 21 was confirmed in the mouse SmartCube® platform and classical forced swim tests, supporting the potential use of α4ß2 partial agonists for treatment of depression.

Synthesis and Behavioral Studies of Chiral Cyclopropanes as Selective α4ß2-Nicotinic Acetylcholine Receptor Partial Agonists Exhibiting an Antidepressant Profile. Part III.

We report the synthesis and biological characterization of novel derivatives of 3-[(1-methyl-2(S)-pyrrolidinyl)methoxy]-5-cyclopropylpyridine (4a-f and 5) as potent and highly selective α4ß2-nicotinic acetylcholine receptor (nAChR) full or partial agonists. A systematic structure-activity study was carried out on the previously described compound 3b, particularly concerning its (2-methoxyethyl)cyclopropyl side-chain, in an effort to improve its metabolic stability while maintaining receptor selectivity. Compound 4d exhibited very similar subnanomolar binding affinity for α4ß2- and α4ß2*-nAChRs compared to 3b, and it showed excellent potency in activating high-sensitivity (HS) α4ß2-nAChRs with an EC50 value of 8.2 nM. Testing of 4d in the SmartCube assay revealed that the compound has a combined antidepressant plus antipsychotic signature. In the forced swim test at a dose of 30 mg/kg given intraperitoneally, 4d was found to be as efficacious as sertraline, thus providing evidence of the potential use of the compound as an antidepressant. Additional promise for use of 4d in humans comes from pharmacokinetic studies in mice indicating brain penetration, and additional assays show compound stability in the presence of human microsomes and hepatocytes. Thus, 4d has a very favorable preclinical drug profile.

Emamectin is a non-selective allosteric activator of nicotinic acetylcholine receptors and GABAA/C receptors.

Avermectins are a group of compounds isolated from a soil-dwelling bacterium. They have been widely used as parasiticides and insecticides, acting by relatively irreversible activation of invertebrate chloride channels. Emamectin is a soluble derivative of an avermectin. It is an insecticide, which persistently activates glutamate-gated chloride channels. However, its effects on mammalian ligand-gated ion channels are unknown. To this end, we tested the effect of emamectin on two cation selective nicotinic receptors and two GABA-gated chloride channels expressed in Xenopus oocytes using two-electrode voltage clamp. Our results demonstrate that emamectin could directly activate α7 nAChR, α4ß2 nAChR, α1ß2γ2 GABAA receptor and ρ1 GABAC receptor concentration dependently, with similar potencies for each channel. However, the potencies for it to activate these channels were at least two orders of magnitude lower than its potency of activating invertebrate glutamate-gated chloride channel. In contrast, ivermectin only activated the α1ß2γ2 GABAA receptor.

Distinctive effects of nicotinic receptor intracellular-loop mutations associated with nocturnal frontal lobe epilepsy.

Previously characterized nicotinic acetylcholine receptor (nAChR) autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE)-associated mutations are found in α2, α4 and ß2 subunit transmembrane (TM) domains. They predominantly increase ACh potency and, for ß2-subunit mutants, increase macroscopic currents. Two recently-identified mutations, α4(R336H) and ß2(V337G), located in the intracellular cytoplasmic loop (C2) have been associated with non-familial NFLE. Effects of these mutations on α4ß2-nAChR function and expression were studied for the first time, using two-electrode voltage clamp recordings in Xenopus laevis oocytes. Biased-ratio preparations elucidated the mutations' effects at alternate isoforms: high-sensitivity [HS; (α4)2(ß2)3] or low-sensitivity [LS; (α4)3(ß2)2] via 1:10 or 30:1 [α4:ß2] cRNA injection ratios, respectively. An unbiased (1:1 [α4:ß2] cRNA) injection ratio was also used to study potential shifts in isoform expression. α4(R336H)-containing receptors showed significant increases in maximal ACh-induced currents (Imax) in all preparations (140% increase compared to wild type control). ß2(V337G)-containing receptors significantly increased Imax in the LS-favoring preparation (20% increase compared to control). Expression of either mutation consistently produced enrichment of HS-isoform expression in all preparations. α4ß2-nAChR harboring either NFLE mutant subunit showed unchanged ACh, sazetidine-A, nicotine, cytisine and mecamylamine potency. However, both mutant subunits enhanced partial agonist efficacies in the LS-biased preparation. Using ß2-subunit-specific [(125)I]mAb 295 immunolabeling, nAChR cell-surface expression was determined. Antibody binding studies revealed that the ß2(V337G) mutation tended to reduce cell-surface expression, and function per receptor was significantly increased by either NFLE mutant subunit in HS-favoring preparations. These findings identify both common and differing features between TM- and C2-domain AD/NFLE-associated mutations. As we discuss, the shared features may be particularly salient to AD/NFLE etiology.

Differential α4(+)/(-)ß2 Agonist-binding Site Contributions to α4ß2 Nicotinic Acetylcholine Receptor Function within and between Isoforms.

Two α4ß2 nicotinic acetylcholine receptor (α4ß2-nAChR) isoforms exist with (α4)2(ß2)3 and (α4)3(ß2)2 subunit stoichiometries and high versus low agonist sensitivities (HS and LS), respectively. Both isoforms contain a pair of α4(+)/(-)ß2 agonist-binding sites. The LS isoform also contains a unique α4(+)/(-)α4 site with lower agonist affinity than the α4(+)/(-)ß2 sites. However, the relative roles of the conserved α4(+)/(-)ß2 agonist-binding sites in and between the isoforms have not been studied. We used a fully linked subunit concatemeric nAChR approach to express pure populations of HS or LS isoform α4ß2*-nAChR. This approach also allowed us to mutate individual subunit interfaces, or combinations thereof, on each isoform background. We used this approach to systematically mutate a triplet of ß2 subunit (-)-face E-loop residues to their non-conserved α4 subunit counterparts or vice versa (ß2HQT and α4VFL, respectively). Mutant-nAChR constructs (and unmodified controls) were expressed in Xenopus oocytes. Acetylcholine concentration-response curves and maximum function were measured using two-electrode voltage clamp electrophysiology. Surface expression was measured with (125)I-mAb 295 binding and was used to define function/nAChR. If the α4(+)/(-)ß2 sites contribute equally to function, making identical ß2HQT substitutions at either site should produce similar functional outcomes. Instead, highly differential outcomes within the HS isoform, and between the two isoforms, were observed. In contrast, α4VFL mutation effects were very similar in all positions of both isoforms. Our results indicate that the identity of subunits neighboring the otherwise equivalent α4(+)/(-)ß2 agonist sites modifies their contributions to nAChR activation and that E-loop residues are an important contributor to this neighbor effect.

Functional Impact of 14 Single Nucleotide Polymorphisms Causing Missense Mutations of Human α7 Nicotinic Receptor.

The α7nicotinic receptor (nAChR) is a major subtype of the nAChRs in the central nervous system, and the receptor plays an important role in brain function. In the dbSNP database, there are 55 single nucleotide polymorphisms (SNPs) that cause missense mutations of the human α7nAChR in the coding region. In this study, we tested the impact of 14 SNPs that cause missense mutations in the agonist binding site or the coupling region between binding site and channel gate on the receptor function. The wild type or mutant receptors were expressed or co-expressed in Xenopus oocytes, and the agonist-induced currents were tested using two-electrode voltage clamp. Our results demonstrated that 6 mutants were nonfunctional, 4 mutants had reduced current expression, and 1 mutants altered ACh and nicotine efficacy in the opposite direction, and one additional mutant had slightly reduced agonist sensitivity. Interestingly, the function of most of these nonfunctional mutants could be rescued by α7nAChR positive allosteric modulator PNU-120596 and agonist-PAM 4BP-TQS. Finally, when coexpressed with the wild type, the nonfunctional mutants could also influence the receptor function. These changes of the receptor properties by the mutations could potentially have an impact on the physiological function of the α7nAChR-mediated cholinergic synaptic transmission and anti-inflammatory effects in the human SNP carriers. Rescuing the nonfunctional mutants could provide a novel way to treat the related disorders.

Resistance to Inhibitors of Cholinesterase 3 (Ric-3) Expression Promotes Selective Protein Associations with the Human α7-Nicotinic Acetylcholine Receptor Interactome.

The α7-nicotinic acetylcholine receptor (α7-nAChR) is a ligand-gated ion channel widely expressed in vertebrates and is associated with numerous physiological functions. As transmembrane ion channels, α7-nAChRs need to be expressed on the surface of the plasma membrane to function. The receptor has been reported to associate with proteins involved with receptor biogenesis, modulation of receptor properties, as well as intracellular signaling cascades and some of these associated proteins may affect surface expression of α7-nAChRs. The putative chaperone resistance to inhibitors of cholinesterase 3 (Ric-3) has been reported to interact with, and enhance the surface expression of, α7-nAChRs. In this study, we identified proteins that associate with α7-nAChRs when Ric-3 is expressed. Using α-bungarotoxin (α-bgtx), we isolated and compared α7-nAChR-associated proteins from two stably transfected, human tumor-derived cell lines: SH-EP1-hα7 expressing human α7-nAChRs and the same cell line further transfected to express Ric-3, SH-EP1-hα7-Ric-3. Mass spectrometric analysis of peptides identified thirty-nine proteins that are associated with α7-nAChRs only when Ric-3 was expressed. Significantly, and consistent with reports of Ric-3 function in the literature, several of the identified proteins are involved in biological processes that may affect nAChR surface expression such as post-translational processing of proteins, protein trafficking, and protein transport. Additionally, proteins affecting the cell cycle, the cytoskeleton, stress responses, as well as cyclic AMP- and inositol triphosphate-dependent signaling cascades were identified. These results illuminate how α-bgtx may be used to isolate and identify α7-nAChRs as well as how the expression of chaperones such as Ric-3 can influence proteins associating with α7-nAChRs. These associating proteins may alter activities of α7-nAChRs to expand their functionally-relevant repertoire as well as to affect biogenesis and membrane trafficking of α7-nAChRs.