A Methyl Scan of the Pyrrolidinium Ring of Nicotine Reveals Significant Differences in Its Interactions with α7 and α4ß2 Nicotinic Acetylcholine Receptors.

The two major nicotinic acetylcholine receptors (nAChRs) in the brain are the α4ß2 and α7 subtypes. A "methyl scan" of the pyrrolidinium ring was used to detect differences in nicotine's interactions with these two receptors. Each methylnicotine was investigated using voltage-clamp and radioligand binding techniques. Methylation at each ring carbon elicited unique changes in nicotine's receptor interactions. Replacing the 1'-N-methyl with an ethyl group or adding a second 1'-N-methyl group significantly reduced interaction with α4ß2 but not α7 receptors. The 2'-methylation uniquely enhanced binding and agonist potency at α7 receptors. Although 3'- and 5'-trans-methylations were much better tolerated by α7 receptors than α4ß2 receptors, 4'-methylation decreased potency and efficacy at α7 receptors much more than at α4ß2 receptors. Whereas cis-5'-methylnicotine lacked agonist activity and displayed a low affinity at both receptors, trans-5'-methylnicotine retained considerable α7 receptor activity. Differences between the two 5'-methylated analogs of the potent pyridyl oxymethylene-bridged nicotine analog A84543 were consistent with what was found for the 5'-methylnicotines. Computer docking of the methylnicotines to the Lymnaea acetylcholine binding protein crystal structure containing two persistent waters predicted most of the changes in receptor affinity that were observed with methylation, particularly the lower affinities of the cis-methylnicotines. The much smaller effects of 1'-, 3'-, and 5'-methylations and the greater effects of 2'- and 4'-methylations on nicotine α7 nAChR interaction might be exploited for the design of new drugs based on the nicotine scaffold. SIGNIFICANCE STATEMENT: Using a comprehensive "methyl scan" approach, we show that the orthosteric binding sites for acetylcholine and nicotine in the two major brain nicotinic acetylcholine receptors interact differently with the pyrrolidinium ring of nicotine, and we suggest reasons for the higher affinity of nicotine for the heteromeric receptor. Potential sites for nicotine structure modification were identified that may be useful in the design of new drugs targeting these receptors.

Discovery of an intrasubunit nicotinic acetylcholine receptor-binding site for the positive allosteric modulator Br-PBTC.

Nicotinic acetylcholine receptor (nAChR) ligands that lack agonist activity but enhance activation in the presence of an agonist are called positive allosteric modulators (PAMs). nAChR PAMs have therapeutic potential for the treatment of nicotine addiction and several neuropsychiatric disorders. PAMs need to be selectively targeted toward certain nAChR subtypes to tap this potential. We previously discovered a novel PAM, (R)-7-bromo-N-(piperidin-3-yl)benzo[b]thiophene-2-carboxamide (Br-PBTC), which selectively potentiates the opening of α4ß2*, α2ß2*, α2ß4*, and (α4ß4)2α4 nAChRs and reactivates some of these subtypes when desensitized (* indicates the presence of other subunits). We located the Br-PBTC-binding site through mutagenesis and docking in α4. The amino acids Glu-282 and Phe-286 near the extracellular domain on the third transmembrane helix were found to be crucial for Br-PBTC's PAM effect. E282Q abolishes Br-PBTC potentiation. Using (α4E282Qß2)2α5 nAChRs, we discovered that the trifluoromethylated derivatives of Br-PBTC can potentiate channel opening of α5-containing nAChRs. Mutating Tyr-430 in the α5 M4 domain changed α5-selectivity among Br-PBTC derivatives. There are two kinds of α4 subunits in α4ß2 nAChRs. Primary α4 forms an agonist-binding site with another ß2 subunit. Accessory α4 forms an agonist-binding site with another α4 subunit. The pharmacological effect of Br-PBTC depends both on its own and agonists' occupancy of primary and accessory α4 subunits. Br-PBTC reactivates desensitized (α4ß2)2α4 nAChRs. Its full efficacy requires intact Br-PBTC sites in at least one accessory and one primary α4 subunit. PAM potency increases with higher occupancy of the agonist sites. Br-PBTC and its derivatives should prove useful as α subunit-selective nAChR PAMs.

Discovery of peptide ligands through docking and virtual screening at nicotinic acetylcholine receptor homology models.

Venom peptide toxins such as conotoxins play a critical role in the characterization of nicotinic acetylcholine receptor (nAChR) structure and function and have potential as nervous system therapeutics as well. However, the lack of solved structures of conotoxins bound to nAChRs and the large size of these peptides are barriers to their computational docking and design. We addressed these challenges in the context of the α4ß2 nAChR, a widespread ligand-gated ion channel in the brain and a target for nicotine addiction therapy, and the 19-residue conotoxin α-GID that antagonizes it. We developed a docking algorithm, ToxDock, which used ensemble-docking and extensive conformational sampling to dock α-GID and its analogs to an α4ß2 nAChR homology model. Experimental testing demonstrated that a virtual screen with ToxDock correctly identified three bioactive α-GID mutants (α-GID[A10V], α-GID[V13I], and α-GID[V13Y]) and one inactive variant (α-GID[A10Q]). Two mutants, α-GID[A10V] and α-GID[V13Y], had substantially reduced potency at the human α7 nAChR relative to α-GID, a desirable feature for α-GID analogs. The general usefulness of the docking algorithm was highlighted by redocking of peptide toxins to two ion channels and a binding protein in which the peptide toxins successfully reverted back to near-native crystallographic poses after being perturbed. Our results demonstrate that ToxDock can overcome two fundamental challenges of docking large toxin peptides to ion channel homology models, as exemplified by the α-GID:α4ß2 nAChR complex, and is extendable to other toxin peptides and ion channels. ToxDock is freely available at rosie.rosettacommons.org/tox_dock.

Investigation of the Possible Pharmacologically Active Forms of the Nicotinic Acetylcholine Receptor Agonist Anabaseine.

Three major forms of the nicotinic agonist toxin anabaseine (cyclic iminium, cyclic imine and the monocationic open-chain ammonium-ketone) co-exist in almost equal concentrations at physiological pH. We asked the question: Which of these forms is pharmacologically active? First, we investigated the pH dependence of anabaseine inhibition of [3H]-methylcarbamylcholine binding at rat brain α4ß2 nicotinic acetylcholine receptors (nAChRs). These experiments indicated that one or both monocationic forms interact with the orthosteric binding site for ACh. However, since they occur at equal concentrations near physiological pH, we employed another approach, preparing a stable analog of each form and examining its agonist activities and binding affinities at several vertebrate brain and neuromuscular nAChRs. Only 2-(3-pyridyl)-1,4,5,6-tetrahydropyrimidine monohydrogen chloride (PTHP), the cyclic iminium analog, displayed nAChR potencies and binding affinities similar to anabaseine. The cyclic imine analog 2,3'-bipyridyl and the open-chain ammonium-ketone analog 5-methylamino-1-(3-pyridyl)-1-pentanone (MAPP), displayed ≤1% of the activity predicted if the one form was solely active. The lower potency of weakly basic 2,3'-bipyridyl can be explained by the presence of a small concentration of its monocationic form. Since the open chain ammonium-ketone monocationic form of anabaseine has some structural similarity to the neurotransmitter GABA, we also tested the ability of anabaseine and its 1,2-dehydropyrrolidinyl analog myosmine to activate a mammalian GABAA receptor, but no activity was detected. We conclude that the monocationic cyclic iminium is the form which avidly binds and activates vertebrate nAChRs.

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.

Unorthodox Acetylcholine Binding Sites Formed by α5 and ß3 Accessory Subunits in α4ß2* Nicotinic Acetylcholine Receptors.

All nicotinic acetylcholine receptors (nAChRs) evolved from homomeric nAChRs in which all five subunits are involved in forming acetylcholine (ACh) binding sites at their interfaces. Heteromeric α4ß2* nAChRs typically have two ACh binding sites at α4/ß2 interfaces and a fifth accessory subunit surrounding the central cation channel. ß2 accessory subunits do not form ACh binding sites, but α4 accessory subunits do at the α4/α4 interface in (α4ß2)2α4 nAChRs. α5 and ß3 are closely related subunits that had been thought to act only as accessory subunits and not take part in forming ACh binding sites. The effect of agonists at various subunit interfaces was determined by blocking homologous sites at these interfaces using the thioreactive agent 2-((trimethylammonium)ethyl) methanethiosulfonate (MTSET). We found that α5/α4 and ß3/α4 interfaces formed ACh binding sites in (α4ß2)2α5 and (α4ß2)2ß3 nAChRs. The α4/α5 interface in (ß2α4)2α5 nAChRs also formed an ACh binding site. Blocking of these sites with MTSET reduced the maximal ACh evoked responses of these nAChRs by 30-50%. However, site-selective agonists NS9283 (for the α4/α4 site) and sazetidine-A (for the α4/ß2 site) did not act on the ACh sites formed by the α5/α4 or ß3/α4 interfaces. This suggests that unorthodox sites formed by α5 and ß3 subunits have unique ligand selectivity. Agonists or antagonists for these unorthodox sites might be selective and effective drugs for modulating nAChR function to treat nicotine addiction and other disorders.

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.

A Novel α2/α4 Subtype-selective Positive Allosteric Modulator of Nicotinic Acetylcholine Receptors Acting from the C-tail of an α Subunit.

Positive allosteric modulators (PAMs) of nicotinic acetylcholine receptors (nAChR) are important therapeutic candidates as well as valuable research tools. We identified a novel type II PAM, (R)-7-bromo-N-(piperidin-3-yl)benzo[b]thiophene-2-carboxamide (Br-PBTC), which both increases activation and reactivates desensitized nAChRs. This compound increases acetylcholine-evoked responses of α2* and α4* nAChRs but is without effect on α3* or α6* nAChRs (* indicates the presence of other nAChR subunits). Br-BPTC acts from the C-terminal extracellular sequences of α4 subunits, which is also a PAM site for steroid hormone estrogens such as 17ß-estradiol. Br-PBTC is much more potent than estrogens. Like 17ß-estradiol, the non-steroid Br-PBTC only requires one α4 subunit to potentiate nAChR function, and its potentiation is stronger with more α4 subunits. This feature enables Br-BPTC to potentiate activation of (α4ß2)(α6ß2)ß3 but not (α6ß2)2ß3 nAChRs. Therefore, this compound is potentially useful in vivo for determining functions of different α6* nAChR subtypes. Besides activation, Br-BPTC affects desensitization of nAChRs induced by sustained exposure to agonists. After minutes of exposure to agonists, Br-PBTC reactivated short term desensitized nAChRs that have at least two α4 subunits but not those with only one. Three α4 subunits were required for Br-BPTC to reactivate long term desensitized nAChRs. These data suggest that higher PAM occupancy promotes channel opening more efficiently and overcomes short and long term desensitization. This C-terminal extracellular domain could be a target for developing subtype or state-selective drugs for nAChRs.

An Accessory Agonist Binding Site Promotes Activation of α4ß2* Nicotinic Acetylcholine Receptors.

Neuronal nicotinic acetylcholine receptors containing α4, ß2, and sometimes other subunits (α4ß2* nAChRs) regulate addictive and other behavioral effects of nicotine. These nAChRs exist in several stoichiometries, typically with two high affinity acetylcholine (ACh) binding sites at the interface of α4 and ß2 subunits and a fifth accessory subunit. A third low affinity ACh binding site is formed when this accessory subunit is α4 but not if it is ß2. Agonists selective for the accessory ACh site, such as 3-[3-(3-pyridyl)-1,2,4-oxadiazol-5-yl]benzonitrile (NS9283), cannot alone activate a nAChR but can facilitate more efficient activation in combination with agonists at the canonical α4ß2 sites. We therefore suggest categorizing agonists according to their site selectivity. NS9283 binds to the accessory ACh binding site; thus it is termed an accessory site-selective agonist. We expressed (α4ß2)2 concatamers in Xenopus oocytes with free accessory subunits to obtain defined nAChR stoichiometries and α4/accessory subunit interfaces. We show that α2, α3, α4, and α6 accessory subunits can form binding sites for ACh and NS9283 at interfaces with α4 subunits, but ß2 and ß4 accessory subunits cannot. To permit selective blockage of the accessory site, α4 threonine 126 located on the minus side of α4 that contributes to the accessory site, but not the α4ß2 sites, was mutated to cysteine. Alkylation of this cysteine with a thioreactive reagent blocked activity of ACh and NS9283 at the accessory site. Accessory agonist binding sites are promising drug targets.

Antigen-specific immunotherapeutic vaccine for experimental autoimmune myasthenia gravis.

Myasthenia gravis (MG) and experimental autoimmune myasthenia gravis (EAMG) are caused by Ab-mediated autoimmune responses to muscle nicotinic acetylcholine receptors (AChRs) that impair neuromuscular transmission, thereby causing muscle weakness. Previously, we discovered that i.p. injection of a therapeutic vaccine consisting of bacterially expressed cytoplasmic domains of human AChR subunits reduced the development of chronic EAMG in rats. In this article, we show that immunization with the therapeutic vaccine in adjuvants does not induce EAMG and, thus, is safe. The potency and efficacy of the therapeutic vaccine were greatly increased by s.c. administration of repeated low doses in IFA. Onset of chronic EAMG could be prevented. Established chronic EAMG could be rapidly reversed, modeling therapy of chronic MG. Therapy reduced pathological Abs assayed by immune precipitation of a main immunogenic region chimera. Successfully treated rats exhibited long-term resistance to reinduction of EAMG, suggesting a lasting cure of MG. A long-term effect of therapy was to change the isotype of the pathogenic Ab response from IgG2b, which fixes complement, to IgG1, which does not. Prevention and reversal of chronic EAMG was not caused by the isotype switch, but the isotype switch may contribute to resistance to reinduction of EAMG. Immunization with AChR cytoplasmic domains in adjuvant is promising as a safe, Ag-specific, potent, effective, rapidly acting, and long-lasting therapeutic approach to MG.

α-Conotoxins Identify the α3ß4* Subtype as the Predominant Nicotinic Acetylcholine Receptor Expressed in Human Adrenal Chromaffin Cells.

Ligands that selectively inhibit human α3ß2 and α6ß2 nicotinic acetylcholine receptor (nAChRs) and not the closely related α3ß4 and α6ß4 subtypes are lacking. Current α-conotoxins (α-Ctxs) that discriminate among these nAChR subtypes in rat fail to discriminate among the human receptor homologs. In this study, we describe the development of α-Ctx LvIA(N9R,V10A) that is 3000-fold more potent on oocyte-expressed human α3ß2 than α3ß4 and 165-fold more potent on human α6/α3ß2ß3 than α6/α3ß4 nAChRs. This analog was used in conjuction with three other α-Ctx analogs and patch-clamp electrophysiology to characterize the nAChR subtypes expressed by human adrenal chromaffin cells. LvIA(N9R,V10A) showed little effect on the acetylcholine-evoked currents in these cells at concentrations expected to inhibit nAChRs with ß2 ligand-binding sites. In contrast, the ß4-selective α-Ctx BuIA(T5A,P6O) inhibited >98% of the acetylcholine-evoked current, indicating that most of the heteromeric receptors contained ß4 ligand-binding sites. Additional studies using the α6-selective α-Ctx PeIA(A7V,S9H,V10A,N11R,E14A) indicated that the predominant heteromeric nAChR expressed by human adrenal chromaffin cells is the α3ß4* subtype (asterisk indicates the possible presence of additional subunits). This conclusion was supported by polymerase chain reaction experiments of human adrenal medulla gland and of cultured human adrenal chromaffin cells that demonstrated prominent expression of RNAs for α3, α5, α7, ß2, and ß4 subunits and a low abundance of RNAs for α2, α4, α6, and α10 subunits.

Chronic sazetidine-A maintains anxiolytic effects and slower weight gain following chronic nicotine without maintaining increased density of nicotinic receptors in rodent brain.

Chronic nicotine administration increases the density of brain α4ß2* nicotinic acetylcholine receptors (nAChRs), which may contribute to nicotine addiction by exacerbating withdrawal symptoms associated with smoking cessation. Varenicline, a smoking cessation drug, also increases these receptors in rodent brain. The maintenance of this increase by varenicline as well as nicotine replacement may contribute to the high rate of relapse during the first year after smoking cessation. Recently, we found that sazetidine-A (saz-A), a potent partial agonist that desensitizes α4ß2* nAChRs, does not increase the density of these receptors in brain at doses that decrease nicotine self-administration, increase attention in rats, and produce anxiolytic effects in mice. Here, we investigated whether chronic saz-A and varenicline maintain the density of nAChRs after their up-regulation by nicotine. In addition, we examined the effects of these drugs on a measure of anxiety in mice and weight gain in rats. After increasing nAChRs in the rodent brain with chronic nicotine, replacing nicotine with chronic varenicline maintained the increased nAChR binding, as well as the α4ß2 subunit proteins measured by western blots. In contrast, replacing nicotine treatments with chronic saz-A resulted in the return of the density of nAChRs to the levels seen in saline controls. Nicotine, saz-A and varenicline each demonstrated anxiolytic effects in mice, but only saz-A and nicotine attenuated the gain of weight over a 6-week period in rats. These findings suggest that apart from its modest anxiolytic and weight control effects, saz-A, or drugs like it, may be useful in achieving long-term abstinence from smoking.

The unique α4+/-α4 agonist binding site in (α4)3(ß2)2 subtype nicotinic acetylcholine receptors permits differential agonist desensitization pharmacology versus the (α4)2(ß2)3 subtype.

Selected nicotinic agonists were used to activate and desensitize high-sensitivity (HS) (α4)2(ß2)3) or low-sensitivity (LS) (α4)3(ß2)2) isoforms of human α4ß2-nicotinic acetylcholine receptors (nAChRs). Function was assessed using (86)Rb(+) efflux in a stably transfected SH-EP1-hα4ß2 human epithelial cell line, and two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes expressing concatenated pentameric HS or LS α4ß2-nAChR constructs (HSP and LSP). Unlike previously studied agonists, desensitization by the highly selective agonists A-85380 [3-(2(S)-azetidinylmethoxy)pyridine] and sazetidine-A (Saz-A) preferentially reduced α4ß2-nAChR HS-phase versus LS-phase responses. The concatenated-nAChR experiments confirmed that approximately 20% of LS-isoform acetylcholine-induced function occurs in an HS-like phase, which is abolished by Saz-A preincubation. Six mutant LSPs were generated, each targeting a conserved agonist binding residue within the LS-isoform-only α4(+)/(-)α4 interface agonist binding site. Every mutation reduced the percentage of LS-phase function, demonstrating that this site underpins LS-phase function. Oocyte-surface expression of the HSP and each of the LSP constructs was statistically indistinguishable, as measured using ß2-subunit-specific [(125)I]mAb295 labeling. However, maximum function is approximately five times greater on a "per-receptor" basis for unmodified LSP versus HSP α4ß2-nAChRs. Thus, recruitment of the α4(+)/(-)α4 site at higher agonist concentrations appears to augment otherwise-similar function mediated by the pair of α4(+)/(-)ß2 sites shared by both isoforms. These studies elucidate the receptor-level differences underlying the differential pharmacology of the two α4ß2-nAChR isoforms, and demonstrate that HS versus LS α4ß2-nAChR activity can be selectively manipulated using pharmacological approaches. Since α4ß2 nAChRs are the predominant neuronal subtype, these discoveries likely have significant functional implications, and may provide important insights for drug discovery and development.

Synthesis and activity of substituted heteroaromatics as positive allosteric modulators for α4ß2α5 nicotinic acetylcholine receptors.

The design and synthesis of a series of substituted heteroaromatic α4ß2α5 positive allosteric modulators is reported. The optimization and development of the heteroaromatic series was carried out from NS9283, and several potent analogues, such as 3-(5-(pyridin-3-yl)-2H-tetrazol-2-yl)benzonitrile (5k) and 3,3'-(2H-tetrazole-2,5-diyl)dipyridine (12 h) with good in vitro efficacy were discovered.

Neuronal nicotinic acetylcholine receptor antibodies in autoimmune central nervous system disorders.

Background: Neuronal nicotinic acetylcholine receptors (nAChRs) are abundant in the central nervous system (CNS), playing critical roles in brain function. Antigenicity of nAChRs has been well demonstrated with antibodies to ganglionic AChR subtypes (i.e., subunit α3 of α3ß4-nAChR) and muscle AChR autoantibodies, thus making nAChRs candidate autoantigens in autoimmune CNS disorders. Antibodies to several membrane receptors, like NMDAR, have been identified in autoimmune encephalitis syndromes (AES), but many AES patients have yet to be unidentified for autoantibodies. This study aimed to develop of a cell-based assay (CBA) that selectively detects potentially pathogenic antibodies to subunits of the major nAChR subtypes (α4ß2- and α7-nAChRs) and its use for the identification of such antibodies in "orphan" AES cases. Methods: The study involved screening of sera derived from 1752 patients from Greece, Turkey and Italy, who requested testing for AES-associated antibodies, and from 1203 "control" patients with other neuropsychiatric diseases, from the same countries or from Germany. A sensitive live-CBA with α4ß2-or α7-nAChR-transfected cells was developed to detect antibodies against extracellular domains of nAChR major subunits. Flow cytometry (FACS) was performed to confirm the CBA findings and indirect immunohistochemistry (IHC) to investigate serum autoantibodies' binding to rat brain tissue. Results: Three patients were found to be positive for serum antibodies against nAChR α4 subunit by CBA and the presence of the specific antibodies was quantitatively confirmed by FACS. We detected specific binding of patient-derived serum anti-nAChR α4 subunit antibodies to rat cerebellum and hippocampus tissue. No serum antibodies bound to the α7-nAChR-transfected or control-transfected cells, and no control serum antibodies bound to the transfected cells. All patients positive for serum anti-nAChRs α4 subunit antibodies were negative for other AES-associated antibodies. All three of the anti-nAChR α4 subunit serum antibody-positive patients fall into the AES spectrum, with one having Rasmussen encephalitis, another autoimmune meningoencephalomyelitis and another being diagnosed with possible autoimmune encephalitis. Conclusion: This study lends credence to the hypothesis that the major nAChR subunits are autoimmune targets in some cases of AES and establishes a sensitive live-CBA for the identification of such patients.

Choline transporter-like protein 4 (CTL4) links to non-neuronal acetylcholine synthesis.

Synthesis of acetylcholine (ACh) by non-neuronal cells is now well established and plays diverse physiologic roles. In neurons, the Na(+) -dependent, high affinity choline transporter (CHT1) is absolutely required for ACh synthesis. In contrast, some non-neuronal cells synthesize ACh in the absence of CHT1 indicating a fundamental difference in ACh synthesis compared to neurons. The aim of this study was to identify choline transporters, other than CHT1, that play a role in non-neuronal ACh synthesis. ACh synthesis was studied in lung and colon cancer cell lines focusing on the choline transporter-like proteins, a five gene family choline-transporter like protein (CTL)1-5. Supporting a role for CTLs in choline transport in lung cancer cells, choline transport was Na(+) -independent and CTL1-5 were expressed in all cells examined. CTL1, 2, and 5 were expressed at highest levels and knockdown of CTL1, 2, and 5 decreased choline transport in H82 lung cancer cells. Knockdowns of CTL1, 2, 3, and 5 had no effect on ACh synthesis in H82 cells. In contrast, knockdown of CTL4 significantly decreased ACh secretion by both lung and colon cancer cells. Conversely, increasing expression of CTL4 increased ACh secretion. These results indicate that CTL4 mediates ACh synthesis in non-neuronal cell lines and presents a mechanism to target non-neuronal ACh synthesis without affecting neuronal ACh synthesis.

α-Conotoxin PeIA[S9H,V10A,E14N] potently and selectively blocks α6ß2ß3 versus α6ß4 nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptors (nAChRs) containing α6 and ß2 subunits modulate dopamine release in the basal ganglia and are therapeutically relevant targets for treatment of neurological and psychiatric disorders including Parkinson's disease and nicotine dependence. However, the expression profile of ß2 and ß4 subunits overlap in a variety of tissues including locus ceruleus, retina, hippocampus, dorsal root ganglia, and adrenal chromaffin cells. Ligands that bind α6ß2 nAChRs also potently bind the closely related α6ß4 subtype. To distinguish between these two subtypes, we synthesized novel analogs of a recently described α-conotoxin, PeIA. PeIA is a peptide antagonist that blocks several nAChR subtypes, including α6/α3ß2ß3 and α6/α3ß4 nAChRs, with low nanomolar potency. We systematically mutated PeIA and evaluated the resulting analogs for enhanced potency and/or selectivity for α6/α3ß2ß3 nAChRs expressed in Xenopus oocytes (α6/α3 is a subunit chimera that contains the N-terminal ligand-binding domain of the α6 subunit). On the basis of these results, second-generation analogs were then synthesized. The final analog, PeIA[S9H,V10A,E14N], potently blocked acetylcholine-gated currents mediated by α6/α3ß2ß3 and α6/α3ß4 nAChRs with IC(50) values of 223 pM and 65 nM, respectively, yielding a >290-fold separation between the two subtypes. Kinetic studies of ligand binding to α6/α3ß2ß3 nAChRs yielded a k(off) of 0.096 ± 0.001 min(-1) and a k(on) of 0.23 ± 0.019 min(-1) M(-9). The synthesis of PeIA[S9H,V10A,E14N] demonstrates that ligands can be developed to discriminate between α6ß2 and α6ß4 nAChRs.

Medico-ethical versus biological evaluationism, and the concept of disease.

According to the 'fact-plus-value' model of pathology propounded by K. W. M. Fulford, 'disease' is a value term that ought to reflect a 'balance of values' stemming from patients and doctors and other 'stakeholders' in medical nosology. In the present article I take issue with his linguistic-analytical arguments for why pathological status must be relative to such a kind of medico-ethical normativity. Fulford is right to point out that Boorse and other naturalists are compelled to utilize evaluative terminology when they characterize the nature of diseases and biological dysfunctions. But the relevant 'biofunctional judgements' are no less factual and empirical for that. While it is indeed evaluative to say that biological dysfunctions involve failures to execute naturally selected functions, such judgments are not bound to entail anything about what is good or bad for us, and what should be treated or not. In the last part of the paper I ruminate briefly on the relationship between 'biological evaluationism', on my construal, and descriptions of 'causal biology'. As I note in my conclusion, a strict bioevaluative concept of disease can be valid for every species on earth, and thus be of particular usefulness in general biological contexts.

Novel Cell-Based Assay for Alpha-3 Nicotinic Receptor Antibodies Detects Antibodies Exclusively in Autoimmune Autonomic Ganglionopathy.

BACKGROUND AND OBJECTIVES: Autoantibodies against α3-subunit-containing nicotinic acetylcholine receptors (α3-nAChRs), usually measured by radioimmunoprecipitation assay (RIPA), are detected in patients with autoimmune autonomic ganglionopathy (AAG). However, low α3-nAChR antibody levels are frequently detected in other neurologic diseases with questionable significance. Our objective was to develop a method for the selective detection of the potentially pathogenic α3-nAChR antibodies, seemingly present only in patients with AAG. METHODS: The study involved sera from 55 patients from Greece, suspected for autonomic failure, and 13 patients from Italy diagnosed with autonomic failure, positive for α3-nAChR antibodies by RIPA. In addition, sera from 52 patients with Ca2+ channel or Hu antibodies and from 2,628 controls with various neuroimmune diseases were included. A sensitive live cell-based assay (CBA) with α3-nAChR-transfected cells was developed to detect antibodies against the cell-exposed α3-nAChR domain. RESULTS: Twenty-five patients were found α3-nAChR antibody positive by RIPA. Fifteen of 25 patients were also CBA positive. Of interest, all 15 CBA-positive patients had AAG, whereas all 10 CBA-negative patients had other neurologic diseases. RIPA antibody levels of the CBA-negative sera were low, although our CBA could detect dilutions of AAG sera corresponding to equally low RIPA antibody levels. No serum bound to control-transfected cells, and none of the 2,628 controls was α3-CBA positive. DISCUSSION: This study showed that in contrast to the established RIPA for α3-nAChR antibodies, which at low levels is of moderate disease specificity, our CBA seems AAG specific, while at least equally sensitive with the RIPA. This study provides Class II evidence that α3-nAChR CBA is a specific assay for AAG. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that an α3-nAChR cell-based assay is a more specific assay for AAG than the standard RIPA.

Expression of functional human α6ß2ß3* acetylcholine receptors in Xenopus laevis oocytes achieved through subunit chimeras and concatamers.

α6ß2ß3* acetylcholine receptors (AChRs) on dopaminergic neurons are important targets for drugs to treat nicotine addiction and Parkinson's disease. However, it has not been possible to efficiently express functional α6ß2ß3* AChRs in oocytes or transfected cells. α6/α3 subunit chimeras permit expression of functional AChRs and reveal that parts of the α6 M1 transmembrane domain and large cytoplasmic domain impair assembly. Concatameric subunits permit assembly of functional α6ß2ß3* AChRs with defined subunit compositions and subunit orders. Assembly of accessory subunits is limiting in formation of mature AChRs. A single linker between the ß3 accessory subunit and an α4 or α6 subunit is sufficient to permit assembly of complex ß3-(α4ß2)(α6ß2) or ß3-(α6ß2)(α4ß2) AChRs. Concatameric pentamers such as ß3-α6-ß2-α4-ß2 have been functionally characterized. α6ß2ß3* AChRs are sensitive to activation by drugs used for smoking cessation therapy (nicotine, varenicline, and cytisine) and by sazetidine. All these are partial agonists. (α6ß2)(α4ß2)ß3 AChRs are most sensitive to agonists. (α6ß2)2ß3 AChRs have the greatest Ca²+ permeability. (α4ß2)(α6ß2)ß3 AChRs are most efficiently transported to the cell surface, whereas (α6ß2)2ß3 AChRs are the least efficiently transported. Dopaminergic neurons may have special chaperones for assembling accessory subunits with α6 subunits and for transporting (α6ß2)2ß3 AChRs to the cell surface. Concatameric pentamers and pentamers formed from combinations of trimers, dimers, and monomers exhibit similar properties, indicating that the linkers between subunits do not alter their functional properties. For the first time, these concatamers allow analysis of functional properties of α6ß2ß3* AChRs. These concatamers should enable selection of drugs specific for α6ß2ß3* AChRs.