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.

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.

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.

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.

Roles for N-terminal extracellular domains of nicotinic acetylcholine receptor (nAChR) ß3 subunits in enhanced functional expression of mouse α6ß2ß3- and α6ß4ß3-nAChRs.

Functional heterologous expression of naturally expressed mouse α6*-nicotinic acetylcholine receptors (mα6*-nAChRs; where "*" indicates the presence of additional subunits) has been difficult. Here we expressed and characterized wild-type (WT), gain-of-function, chimeric, or gain-of-function chimeric nAChR subunits, sometimes as hybrid nAChRs containing both human (h) and mouse (m) subunits, in Xenopus oocytes. Hybrid mα6mß4hß3- (∼ 5-8-fold) or WT mα6mß4mß3-nAChRs (∼ 2-fold) yielded higher function than mα6mß4-nAChRs. Function was not detected when mα6 and mß2 subunits were expressed together or in the additional presence of hß3 or mß3 subunits. However, function emerged upon expression of mα6mß2mß3(V9'S)-nAChRs containing ß3 subunits having gain-of-function V9'S (valine to serine at the 9'-position) mutations in transmembrane domain II and was further elevated 9-fold when hß3(V9'S) subunits were substituted for mß3(V9'S) subunits. Studies involving WT or gain-of-function chimeric mouse/human ß3 subunits narrowed the search for domains that influence functional expression of mα6*-nAChRs. Using hß3 subunits as templates for site-directed mutagenesis studies, substitution with mß3 subunit residues in extracellular N-terminal domain loops "C" (Glu(221) and Phe(223)), "E" (Ser(144) and Ser(148)), and "ß2-ß3" (Gln(94) and Glu(101)) increased function of mα6mß2*- (∼ 2-3-fold) or mα6mß4* (∼ 2-4-fold)-nAChRs. EC50 values for nicotine acting at mα6mß4*-nAChR were unaffected by ß3 subunit residue substitutions in loop C or E. Thus, amino acid residues located in primary (loop C) or complementary (loops ß2-ß3 and E) interfaces of ß3 subunits are some of the molecular impediments for functional expression of mα6mß2ß3- or mα6mß4ß3-nAChRs.

Bupropion and bupropion analogs as treatments for CNS disorders.

Bupropion, introduced as an antidepressant in the 1980s, is also effective as a smoking cessation aid and is beneficial in the treatment of methamphetamine addiction, cocaine dependence, addictive behaviors such as pathological gambling, and attention deficit hyperactivity disorder. (2S,3S)-hydroxybupropion is an active metabolite of bupropion produced in humans that contributes to antidepressant and smoking cessation efficacy and perhaps benefits in other CNS disorders. Mechanisms underlying its antidepressant and smoking abstinence remain elusive. However, it seems likely that efficacy is due to a combination of the effects of bupropion and/or its active metabolite (2S,3S)-hydroxybupropion involving the inhibition of reuptake of dopamine (DA) and NE in reward centers of the brain and the noncompetitive antagonism of α4ß2- and α3ß4*-nAChRs. These combined effects of bupropion and its active metabolite may be responsible for its ability to decrease nicotine reward and withdrawal. Studies directed toward development of a bupropion analog for treatment of cocaine addiction led to compounds, typified by 2-(N-cyclopropylamino)-3'-chloropropiophenone (RTI-6037-39), thought to act as indirect DA agonists. In addition, (2S,3S)-hydroxybupropion analogs were developed, which had varying degrees of DA and NE uptake inhibition and antagonism of nAChRs. These compounds will be valuable tools for animal behavioral studies and as clinical candidates. Here, we review the (1) early studies leading to the development of bupropion, (2) bupropion metabolism and the identification of (2S,3R)-hydroxybupropion as an active metabolite, (3) mechanisms of bupropion and metabolite action, (4) effects in animal behavioral studies, (5) results of clinical studies, and (6) development of bupropion analogs as potential pharmacotherapies for treating nicotine and cocaine addiction.

A signal peptide missense mutation associated with nicotine dependence alters α2*-nicotinic acetylcholine receptor function.

A cytosine to thymidine (C â†’ T) missense mutation in the signal peptide (SP) sequence (rs2472553) of the nicotinic acetylcholine receptor (nAChR) α2 subunit produces a threonine-to-isoleucine substitution (T22I) often associated with nicotine dependence (ND). We assessed effects on function of α2*-nAChR ('*'indicates presence of additional subunits) of this mutation, which could alter SP cleavage, RNA/protein secondary structure, and/or efficiency of transcription, translation, subunit assembly, receptor trafficking or cell surface expression. Two-electrode voltage clamp analyses indicate peak current responses to ACh or nicotine are decreased 2.8-5.8-fold for putative low sensitivity (LS; 10:1 ratio of α:ß subunit cRNAs injected) α2ß2- or α2ß4-nAChR and increased for putative high sensitivity (HS; 1:10 α:ß subunit ratio) α2ß2- (5.7-15-fold) or α2ß4- (1.9-2.2-fold) nAChR as a result of the mutation. Agonist potencies are decreased 1.6-4-fold for putative LS or HS α2(T22I)ß2-nAChR or for either α2*-nAChR subtype formed in the presence of equal amounts of subunit cRNA, slightly decreased for LS α2(T22I)ß4-nAChR, but increased 1.4-2.4-fold for HS α2(T22I)ß4-nAChR relative to receptors containing wild-type α2 subunits. These effects suggest that the α2 subunit SP mutation generally favors formation of LS receptor isoforms. We hypothesize that lower sensitivity of human α2*-nAChR to nicotine could contribute to increased susceptibility to ND. To our knowledge this is the first report of a SP mutation having a functional effect in a member of cys-loop family of ligand-gated ion channels.

Chemistry, pharmacology, and behavioral studies identify chiral cyclopropanes as selective α4ß2-nicotinic acetylcholine receptor partial agonists exhibiting an antidepressant profile. Part II.

A 3-pyridyl ether scaffold bearing a cyclopropane-containing side chain was recently identified in our efforts to create novel antidepressants that act as partial agonists at α4ß2-nicotinic acetylcholine receptors. In this study, a systematic structure-activity relationship investigation was carried out on both the azetidine moiety present in compound 3 and its right-hand side chain, thereby discovering a variety of novel nicotinic ligands that retain bioactivity and feature improved chemical stability. The most promising compounds, 24, 26, and 30, demonstrated comparable or enhanced pharmacological profiles compared to the parent compound 4, and the N-methylpyrrolidine analogue 26 also exhibited robust antidepressant-like efficacy in the mouse forced swim test. The favorable ADMET profile and chemical stability of 26 further indicate this compound to be a promising lead as a drug candidate warranting further advancement down the drug discovery pipeline.

Differential modulation of EAE by α9*- and ß2*-nicotinic acetylcholine receptors.

Nicotine is a potent inhibitor of the immune response and is protective against experimental autoimmune encephalomyelitis (EAE). Initial studies suggested that the cholinergic system modulates inflammation via the α7-nicotinic acetylcholine receptor (nAChR) subtype. We recently have shown that effector T cells and myeloid cells constitutively express mRNAs encoding nAChR α9 and ß2 subunits and found evidence for immune system roles for non-α7-nAChRs. In the present study, we assessed the effects of nAChR α9 or ß2 subunit gene deletion on EAE onset and severity, with or without nicotine treatment. We report again that disease onset is delayed and severity is attenuated in nicotine-treated, wild-type mice, an effect that also is observed in α9 subunit knock-out (KO) mice irrespective of nicotine treatment. On the other hand, ß2 KO mice fail to recover from peak measures of disease severity regardless of nicotine treatment, despite retaining sensitivity to nicotine's attenuation of disease severity. Prior to disease onset, we found significantly less reactive oxygen species production in the central nervous system (CNS) of ß2 KO mice, elevated proportions of CNS myeloid cells but decreased ratios of CNS macrophages/microglia in α9 or ß2 KO mice, and some changes in iNOS, TNF-α and IL-1ß mRNA levels in α9 KO and/or ß2 KO mice. Our data thus suggest that ß2*- and α9*-nAChRs, in addition to α7-nAChRs, have different roles in endogenous and nicotine-dependent modulation of immune functions and could be exploited as therapeutic targets to modulate inflammation and autoimmunity.

Nicotine-morphine interactions at α4ß2, α7 and α3(⁎) nicotinic acetylcholine receptors.

Nicotine and opioids share several behavioral and rewarding properties. Although both opioids and nicotine have their own specific mechanism of action, there is empirical and experimental evidence of interactions between these drugs. We studied receptor-level interactions of nicotine and morphine at α4ß2, α7 and α3(⁎) nicotinic acetylcholine receptors. [(3)H]epibatidine displacement was used to determine if morphine binds competitively to nicotinic acetylcholine receptors. Functional interactions of morphine and nicotine were studied with calcium fluorometry and (86)Rb(+) efflux assays. Morphine displaced [(3)H]epibatidine from nicotinic agonist binding sites in all cell lines studied. The Ki values for morphine were 13.2µM in SH-EP1-hα4ß2 cells, 0.16µM and 126µM in SH-SY5Y cells and 43.7µM in SH-EP1-hα7 cells. In SH-EP1-hα4ß2 cells expressing α4ß2 nicotinic acetylcholine receptors, morphine acted as a partial agonist of (86)Rb(+) efflux comparable to cytisine (with EC50 values of 53.3µM for morphine and 5.38µM for cytisine). The effect of morphine was attenuated concentration-dependently by the nicotinic antagonist mecamylamine. In the SH-SY5Y cell line expressing several subtypes of nicotinic acetylcholine receptors morphine had an inhibitory effect on nicotine induced (86)Rb(+) ion efflux mediated by α3(⁎) nicotinic acetylcholine receptors. These results suggest that morphine acts as a partial agonist at α4ß2 nicotinic acetylcholine receptors and as a weak antagonist at α3(⁎) nicotinic acetylcholine receptors.

Discovery of highly potent and selective α4ß2-nicotinic acetylcholine receptor (nAChR) partial agonists containing an isoxazolylpyridine ether scaffold that demonstrate antidepressant-like activity. Part II.

In our continued efforts to develop α4ß2-nicotinic acetylcholine receptor (nAChR) partial agonists as novel antidepressants having a unique mechanism of action, structure-activity relationship (SAR) exploration of certain isoxazolylpyridine ethers is presented. In particular, modifications to both the azetidine ring present in the starting structure 4 and its metabolically liable hydroxyl side chain substituent have been explored to improve compound druggability. The pharmacological characterization of all new compounds has been carried out using [(3)H]epibatidine binding studies together with functional assays based on (86)Rb(+) ion flux measurements. We found that the deletion of the metabolically liable hydroxyl group or its replacement by a fluoromethyl group not only maintained potency and selectivity but also resulted in compounds showing antidepressant-like properties in the mouse forced swim test. These isoxazolylpyridine ethers appear to represent promising lead candidates in the design of innovative chemical tools containing reporter groups for imaging purposes and of possible therapeutics.

Structure-activity studies of 7-heteroaryl-3-azabicyclo[3.3.1]non-6-enes: a novel class of highly potent nicotinic receptor ligands.

The potential for nicotinic ligands with affinity for the α4ß2 or α7 subtypes to treat such diverse diseases as nicotine addiction, neuropathic pain, and neurodegenerative and cognitive disorders has been exhibited clinically for several compounds while preclinical activity in relevant in vivo models has been demonstrated for many more. For several therapeutic programs, we sought nicotinic ligands with various combinations of affinity and function across both subtypes, with an emphasis on dual α4ß2-α7 ligands, to explore the possibility of synergistic effects. We report here the structure-activity relationships (SAR) for a novel series of 7-heteroaryl-3-azabicyclo[3.3.1]non-6-enes and characterize many of the analogues for activity at multiple nicotinic subtypes.

Impact of prefrontal cortex in nicotine-induced excitation of ventral tegmental area dopamine neurons in anesthetized rats.

Systemic administration of nicotine increases dopaminergic (DA) neuron firing in the ventral tegmental area (VTA), which is thought to underlie nicotine reward. Here, we report that the medial prefrontal cortex (mPFC) plays a critical role in nicotine-induced excitation of VTA DA neurons. In chloral hydrate-anesthetized rats, extracellular single-unit recordings showed that VTA DA neurons exhibited two types of firing responses to systemic nicotine. After nicotine injection, the neurons with type-I response showed a biphasic early inhibition and later excitation, whereas the neurons with type-II response showed a monophasic excitation. The neurons with type-I, but not type-II, response exhibited pronounced slow oscillations (SOs) in firing. Pharmacological or structural mPFC inactivation abolished SOs and prevented systemic nicotine-induced excitation in the neurons with type-I, but not type-II, response, suggesting that these VTA DA neurons are functionally coupled to the mPFC and nicotine increases firing rate in these neurons in part through the mPFC. Systemic nicotine also increased the firing rate and SOs in mPFC pyramidal neurons. mPFC infusion of a non-α7 nicotinic acetylcholine receptor (nAChR) antagonist mecamylamine blocked the excitatory effect of systemic nicotine on the VTA DA neurons with type-I response, but mPFC infusion of nicotine failed to excite these neurons. These results suggest that nAChR activation in the mPFC is necessary, but not sufficient, for systemic nicotine-induced excitation of VTA neurons. Finally, systemic injection of bicuculline prevented nicotine-induced firing alterations in the neurons with type-I response. We propose that the mPFC plays a critical role in systemic nicotine-induced excitation of VTA DA neurons.

Modulation of recombinant, α2*, α3* or α4*-nicotinic acetylcholine receptor (nAChR) function by nAChR ß3 subunits.

The nicotinic acetylcholine receptor (nAChR) ß3 subunit is thought to serve an accessory role in nAChR subtypes expressed in dopaminergic regions implicated in drug dependence and reward. When ß3 subunits are expressed in excess, they have a dominant-negative effect on function of selected nAChR subtypes. In this study, we show, in Xenopus oocytes expressing α2, α3 or α4 plus either ß2 or ß4 subunits, that in the presumed presence of similar amounts of each nAChR subunit, co-expression with wild-type ß3 subunits generally (except for α3*-nAChR) lowers amplitudes of agonist-evoked, inward peak currents by 20-50% without having dramatic effects (≤ 2-fold) on agonist potencies. By contrast, co-expression with mutant ß3(V9'S) subunits generally (except for α4ß2*-nAChR) increases agonist potencies, consistent with an expected gain-of-function effect. This most dramatically demonstrates formation of complexes containing three kinds of subunit. Moreover, for oocytes expressing nAChR containing any α subunit plus ß4 and ß3(V9'S) subunits, there is spontaneous channel opening sensitive to blockade by the open channel blocker, atropine. Collectively, the results indicate that ß3 subunits integrate into all of the studied receptor assemblies and suggest that natural co-expression with ß3 subunits can influence levels of expression and agonist sensitivities of several nAChR subtypes.

α7ß2 nicotinic acetylcholine receptors assemble, function, and are activated primarily via their α7-α7 interfaces.

We investigated assembly and function of nicotinic acetylcholine receptors (nAChRs) composed of α7 and ß2 subunits. We measured optical and electrophysiological properties of wild-type and mutant subunits expressed in cell lines and Xenopus laevis oocytes. Laser scanning confocal microscopy indicated that fluorescently tagged α7 and ß2 subunits colocalize. Förster resonance energy transfer between fluorescently tagged subunits strongly suggested that α7 and ß2 subunits coassemble. Total internal reflection fluorescence microscopy revealed that assemblies localized to filopodia-like processes of SH-EP1 cells. Gain-of-function α7 and ß2 subunits confirmed that these subunits coassemble within functional receptors. Moreover, α7ß2 nAChRs composed of wild-type subunits or fluorescently tagged subunits had pharmacological properties similar to those of α7 nAChRs, although amplitudes of α7ß2 nAChR-mediated, agonist-evoked currents were generally ~2-fold lower than those for α7 nAChRs. It is noteworthy that α7ß2 nAChRs displayed sensitivity to low concentrations of the antagonist dihydro-ß-erythroidine that was not observed for α7 nAChRs at comparable concentrations. In addition, cysteine mutants revealed that the α7-ß2 subunit interface does not bind ligand in a functionally productive manner, partly explaining lower α7ß2 nAChR current amplitudes and challenges in identifying the function of native α7ß2 nAChRs. On the basis of our findings, we have constructed a model predicting receptor function that is based on stoichiometry and position of ß2 subunits within the α7ß2 nAChRs.

Identification of novel α4ß2-nicotinic acetylcholine receptor (nAChR) agonists based on an isoxazole ether scaffold that demonstrate antidepressant-like activity.

There is considerable evidence to support the hypothesis that the blockade of nAChR is responsible for the antidepressant action of nicotinic ligands. The nicotinic acetylcholine receptor (nAChR) antagonist, mecamylamine, has been shown to be an effective add-on in patients that do not respond to selective serotonin reuptake inhibitors. This suggests that nAChR ligands may address an unmet clinical need by providing relief from depressive symptoms in refractory patients. In this study, a new series of nAChR ligands based on an isoxazole-ether scaffold have been designed and synthesized for binding and functional assays. Preliminary structure-activity relationship (SAR) efforts identified a lead compound 43, which possesses potent antidepressant-like activity (1 mg/kg, IP; 5 mg/kg, PO) in the classical mouse forced swim test. Early stage absorption, distribution, metabolism, excretion, and toxicity (ADME-Tox) studies also suggested favorable drug-like properties, and broad screening toward other common neurotransmitter receptors indicated that compound 43 is highly selective for nAChRs over the other 45 neurotransmitter receptors and transporters tested.

Reporter mutation studies show that nicotinic acetylcholine receptor (nAChR) α5 Subunits and/or variants modulate function of α6*-nAChR.

To further the understanding of functional α6α5*-nicotinic acetylcholine receptors (nAChR; the asterisk (*) indicates known or possible presence of other subunits), we have heterologously expressed in oocytes different, mouse or human, nAChR subunit combinations. Coexpression with wild-type α5 subunits or chimeric α5/ß3 subunits (in which the human α5 subunit N-terminal, extracellular domain is linked to the remaining domains of the human ß3 subunit) almost completely abolishes the very small amount of function seen for α6ß4*-nAChR and does not induce function of α6ß2*-nAChR. Coexpression with human α5(V9)'(S) subunits bearing a valine 290 to serine mutation in the 9' position of the second transmembrane domain does not rescue the function of α6ß4*-nAChR or induce function of α6ß2*-nAChR. However, coexpression with mutant chimeric α5/ß3(V9)'(S) subunits has a gain-of-function effect (higher functional expression and agonist sensitivity and spontaneous opening inhibited by mecamylamine) on α6ß4*-nAChR. Moreover, N143D + M145V mutations in the α6 subunit N-terminal domain enable α5/ß3(V9)'(S) subunits to have a gain-of-function effect on α6ß2*-nAChR. nAChR containing chimeric α6/α3 subunits plus either ß2 or ß4 subunits have some function that is modulated in the presence of α5 or α5/ß3 subunits. Coexpression with α5/ß3(V9)'(S) subunits has a gain-of-function effect more pronounced than that in the presence of α5(V9)'(S) subunits. Gain-of-function effects are dependent, sometimes subtly, on the nature and apparently the extracellular, cytoplasmic, and/or transmembrane domain topology of partner subunits. These studies yield insight into assembly of functional α6α5*-nAChR and provide tools for development of α6*-nAChR-selective ligands that could be important in the treatment of nicotine dependence, and perhaps other neurological diseases.