Potentiation of human alpha4beta2 neuronal nicotinic acetylcholine receptor by estradiol.

The modulation of neurotransmitter receptors by various substances can reflect important physiological mechanisms involved in the regulation of neural function. Furthermore, such substances, in particular specific allosteric modulators, can reveal promising therapeutic targets for diseases of the nervous system. From this perspective, we investigated the effects of the steroid hormone estradiol on human neuronal nicotinic acetylcholine receptors expressed either in Xenopus laevis oocytes or human embryonic kidney cells. Acetylcholine-evoked currents were potentiated both by pre- and coapplications of estradiol in alpha4beta2 and alpha4beta4 receptors, but not in alpha3beta2 or alpha3beta4 receptors. The reversible potentiation of alpha4-containing receptors could be induced within seconds in X. laevis oocytes and at micromolar concentrations of estradiol. The potentiation was greatest for responses evoked by low concentrations of acetylcholine, resulting in an apparent increase of receptor affinity. At the single channel level, estradiol potentiation resulted from an increase in opening probability. Finally, the use of functional chimeric or truncated alpha4 subunits demonstrated that a site at the C-terminal tail of the alpha4 subunit is required for estradiol potentiation. These results suggest the presence of a specific site at the human nicotinic acetylcholine receptor alpha4 subunit through which estradiol can cause an allosteric potentiation of acetylcholine-evoked responses.     

Effects of ginsenosides,active components of ginseng,on nicotinic acetylcholine receptors expressed in Xenopus oocytes

We investigated the effects of ginsenosides, the active ingredient of ginseng, on neuronal or muscle-type nicotinic acetylcholine receptor channel activity expressed in Xenopus oocytes after injection of cRNA encoding bovine neuronal alpha3beta4, alpha7 or human muscle alphabetadeltavarepsilon subunits. Treatment with acetylcholine elicited an inward peak current (I(ACh)) in oocytes expressing nicotinic acetylcholine receptor subtypes. Cotreatment with ginsenoside Rg2 and acetylcholine inhibited I(ACh) in oocytes expressing with alpha3beta4 or alphabetadeltavarepsilon but not in oocytes expressing alpha7 nicotinic acetylcholine receptors. The inhibition of I(ACh) by ginsenoside Rg2 was reversible and dose-dependent. The half-inhibitory concentrations (IC50) of ginsenoside Rg2 were 60.2+/-14.1 and 15.7+/-3.5 microM in oocytes expressing alpha3beta4 and alphabetadeltavarepsilon nicotinic acetylcholine receptors, respectively. The inhibition of I(ACh) by ginsenoside Rg2 was voltage-independent and noncompetitive. Other ginsenosides besides ginsenoside Rg2 also inhibited I(ACh) in oocytes expressing alpha3beta4 or alphabetadeltavarepsilon nicotinic acetylcholine receptors. The order of potency for the inhibition of I(ACh) was ginsenoside Rg2>Rf>Re>Rg1>Rc>Rb2>Rb1 in oocytes expressing alpha3beta4 nicotinic acetylcholine receptors and was ginsenoside Rg2>Rf>Rg1>Re>Rb1>Rc>Rb2 in oocytes expressing alphabetadeltavarepsilon nicotinic acetylcholine receptors. These results indicate that ginsenosides might regulate nicotinic acetylcholine receptors in a differential manner and this regulation might be one of the pharmacological actions of Panax ginseng.     

Formation of functional alpha3beta4alpha5 human neuronal nicotinic receptors in Xenopus oocytes: a reporter mutation approach

1. The alpha5 subunit participates to the formation of native neuronal nicotinic receptors, particularly in autonomic ganglia. Like the related beta3 subunit, alpha5 forms functional recombinant receptors if expressed together with a pair of typical alpha and beta subunits, but its effect on the properties of the resulting alphabetaalpha5 receptor depends on the alpha and beta subunits chosen and on the expression system. We used a reporter mutation approach to test whether alpha5, like beta3, is incorporated as a single copy in human alpha3beta4alpha5 receptors expressed in oocytes. 2. As previously reported, the main indication of the presence of alpha5 in alpha3beta4alpha5(wt) was an increase in apparent receptor desensitization (compared with alpha3beta4 receptors). If the alpha3beta4alpha5 receptor bore a 9'T mutation in the second transmembrane domain of either alpha3 or beta4, alpha5 incorporation produced a decrease in ACh sensitivity (by 4 fold for alpha3(LT)beta4alpha5 vs. alpha3(LT)beta4 and by 40 fold for alpha3beta4(LT)alpha5 vs. alpha3beta4(LT)). The much greater effect observed in alpha3beta4(LT)alpha5 receptors accords with the hypothesis that alpha5 takes the place of a beta subunit in the receptor. 3. Introducing a 9'T mutation in alpha5 had no effect on the agonist sensitivity of alpha3beta4alpha5 receptors, but reduced apparent desensitisation, as judged by the sag in the current response to high agonist concentrations. 4. Introducing the 9'T mutation in alpha3 or beta4 in the triplet receptor reduced the EC(50) for ACh by a similar extent (7 and 9 fold, respectively), suggesting that alpha3beta4alpha5 receptors contain two copies each of alpha and beta and therefore only one copy of alpha5.     

5-I A-85380 and TC-2559 differentially activate heterologously expressed alpha4beta2 nicotinic receptors

The neuronal nicotinic acetylcholine receptor alpha4 and beta2 subunits expressed in heterologous expression systems assemble into at least two distinct subunit stoichiometries of alpha4beta2 receptor. The (alpha4)2(beta2)3 stoichiometry is about 100-fold more sensitive to acetylcholine than the (alpha4)3(beta2)2 stoichiometry. In order to investigate if agonists in general distinguish high- and low-affinity alpha4beta2 nicotinic acetylcholine receptors, we have expressed human alpha4 and beta2 nicotinic acetylcholine receptor subunits in two different expression systems. The relative amounts of alpha4beta2 nicotinic acetylcholine receptors with high- and low-affinity for acetylcholine were manipulated by (a) injecting the subunit cDNAs at different alpha:beta ratios into Xenopus oocytes and (b) by culturing HEK-293 cells stably expressing alpha4beta2 nicotinic acetylcholine receptors overnight at different temperatures. The sensitivities of the alpha4beta2 nicotinic acetylcholine receptors to the agonists acetylcholine, 5-I A-85380, and TC-2559 were investigated using the voltage-clamp technique on Xenopus oocytes and using a fluorescent imaging plate reader to measure calcium responses from HEK-293 cells. Like acetylcholine, 5-I A-85380 produced biphasic concentration-response curves and the high-affinity component became larger when the cells were manipulated to produce a greater proportion of (alpha4)2(beta2)3 nicotinic acetylcholine receptors. Interestingly, under all circumstances, TC-2559 produced monophasic concentration-response curves. In oocytes injected with alpha4 and beta2 subunits in the 1:1 ratio the maximum effect of TC-2559 was 28% of that of acetylcholine. The EC50 for TC-2559 was not changed when oocytes were manipulated to express exclusively (alpha4)2(beta2)3 nicotinic acetylcholine receptors, however, the maximum effect of TC-2559 was dramatically enhanced. These results suggest that TC-2559 is a selective agonist of the (alpha4)2(beta2)3 nicotinic acetylcholine receptor stoichiometry.     

Acetylcholine receptor extracellular domain determines sensitivity to nicotine-induced inactivation

We have shown previously that chronic exposure to submicromolar concentrations of nicotine permanently inactivates alpha4beta2 and alpha7 neuronal nicotinic acetylcholine receptors while alpha3beta2 acetylcholine receptors are resistant to inactivation. Phosphorylation of the large cytoplasmic domain has been proposed to mediate functional inactivation. Chimeric subunits consisting of human alpha4 sequence from their N-terminus to either the beginning of the first transmembrane domain or the large cytoplasmic domain and alpha3 sequences thereafter formed acetylcholine receptors with beta2 subunits which were as susceptible to nicotine-induced inactivation as wild-type alpha4 acetylcholine receptors. The converse chimeras, containing the N-terminal parts of the alpha3 subunit and the C-terminal parts of the alpha4 subunit, formed acetylcholine receptors with beta2 subunits which were as resistant to nicotine-induced inactivation as wild-type alpha3beta2 acetylcholine receptors. Thus, inactivation of acetylcholine receptors produced by chronic exposure to nicotine results primarily from effects of the agonist on the extracellular and transmembrane domains of the alpha subunit.     

A subtype of nicotinic cholinergic receptor in rat brain is composed of alpha 4 and beta 2 subunits and is up-regulated by chronic nicotine treatment.

The subunit composition and pharmacological regulation of rat neuronal nicotinic cholinergic receptors were assessed. Specific immunoprecipitation was determined in solubilized rat brain homogenates using [3H]cytisine, a high affinity agonist at nicotinic receptors, in conjunction with polyclonal antisera generated against nonhomologous domains of the various subunits comprising this receptor class. In all brain regions tested, only antisera generated against the alpha 4 and beta 2 subunits were able to immunoprecipitate specifically receptors labeled by [3H]cytisine. Thus, these sera were further characterized in order to validate and optimize their use in the immunoprecipitation protocol. Preincubation of solubilized receptors from rat forebrain with antisera generated against the alpha 2, alpha 3, alpha 5, beta 3, or beta 4 subunits did not decrease the amount of precipitable alpha 4 or beta 2 subunit. On the other hand, when either anti-alpha 4 or anti-beta 2 serum was used to immunoprecipitate solubilized receptors from rat forebrain, the supernatants contained little if any remaining receptors that could be specifically precipitated by either antibody. Because these antisera do not cross-react, the data indicate that alpha 4 and beta 2 subunits are associated with each other in at least one neuronal nicotinic receptor subtype that has high affinity for agonists. Moreover, these results imply that all alpha 4 subunits that are labeled by [3H]cystisine are coupled to beta 2 subunits. We also present evidence that the alpha 4/beta 2 subtype characterized in this report is significantly increased in the cortex of rats chronically treated with nicotine.     

The effects of subunit composition on the inhibition of nicotinic receptors by the amphipathic blocker 2,2,6,6-tetramethylpiperidin-4-yl heptanoate

The therapeutic targeting of nicotinic receptors in the brain will benefit from the identification of drugs that may be selective for their ability to activate or inhibit a limited range of nicotine acetylcholine receptor subtypes. In the present study, we describe the effects of 2,2,6,6-tetramethylpiperidin-4-yl heptanoate (TMPH), a novel compound that is a potent inhibitor of neuronal nicotinic receptors. Evaluation of nicotinic acetylcholine receptor (nAChR) subunits expressed in Xenopus laevis oocytes indicated that TMPH can produce a potent and long-lasting inhibition of neuronal nAChR formed by the pairwise combination of the most abundant neuronal alpha (i.e., alpha3 and alpha4) and beta subunits (beta2 and beta4), with relatively little effect, because of rapid reversibility of inhibition, on muscle-type (alpha1beta1gammadelta) or alpha7 receptors. However, the inhibition of neuronal beta subunit-containing receptors was also decreased if any of the nonessential subunits alpha5, alpha6, or beta3 were coexpressed. This decrease in inhibition is shown to be associated with a single amino acid present in the second transmembrane domain of these subunits. Our data indicate great potential utility for TMPH to help relate the diverse central nervous system effects to specific nAChR subtypes.     

Novel iboga alkaloid congeners block nicotinic receptors and reduce drug self-administration

18-Methoxycoronaridine, a novel iboga alkaloid congener, reduces drug self-administration in animal models of addiction. Previously, we proposed that these effects are mediated by the ability of 18-methoxycoronaridine to inhibit nicotinic alpha3beta4 acetylcholine receptors. In an attempt to identify more potent 18-methoxycoronaridine analogs, we have tested a series of 18-methoxycoronaridine congeners by whole-cell patch clamp recording of HEK 293 cells expressing recombinant nicotinic alpha3beta4 receptors or glutamate NR1/NR2B N-methyl-d-aspartate (NMDA) receptors. The congeners exhibited a range of inhibitory potencies at alpha3beta4 receptors. Five congeners had IC(50) values similar to 18-methoxycoronaridine, and all of these were ineffective at NMDA receptors. The congeners also retained their ability to reduce morphine and methamphetamine self-administration. These data are consistent with the importance of nicotinic alpha3beta4 receptors as a therapeutic target to modulate drug seeking. These compounds may constitute a new class of synthetic agents that act via the nicotinic alpha3beta4 mechanism to combat addiction.     

Rhesus monkey alpha7 nicotinic acetylcholine receptors: comparisons to human alpha7 receptors expressed in Xenopus oocytes

An alpha7 nicotinic acetylcholine receptor sequence was cloned from Rhesus monkey (Macaca mulatta). This clone differs from the mature human alpha7 nicotinic acetylcholine receptor in only four amino acids, two of which are in the extracellular domain. The monkey alpha7 nicotinic receptor was characterized in regard to its functional responses to acetylcholine, choline, cytisine, and the experimental alpha7-selective agonists 4OH-GTS-21, TC-1698, and AR-R17779. For all of these agonists, the EC(50) for activation of monkey receptors was uniformly higher than for human receptors. In contrast, the potencies of mecamylamine and MLA for inhibiting monkey and human alpha7 were comparable. Acetylcholine and 4OH-GTS-21 were used to probe the significance of the single point differences in the extracellular domain. Mutants with the two different amino acids in the extracellular domain of the monkey receptor changed to the corresponding sequence of the human receptor had responses to these agonists that were not significantly different in EC(50) from wild-type human alpha7 nicotinic receptors. Monkey alpha7 nicotinic receptors have a serine at residue 171, while the human receptors have an asparagine at this site. Monkey S171N mutants were more like human alpha7 nicotinic receptors, while mutations at the other site (K186R) had relatively little effect. These experiments point toward the basic utility of the monkey receptor as a model for the human alpha7 nicotinic receptor, albeit with the caveat that these receptors will vary in their agonist concentration dependency. They also point to the potential importance of a newly identified sequence element for modeling the specific amino acids involved with receptor activation.     

Effects of the plant alkaloid tetrandrine on human nicotinic acetylcholine receptors

Functional effects of the well-characterized antagonist of L-type Ca(2+) channels tetrandrine on recombinant human gamma-aminobutyric acid type A (GABA(A)) (alpha1beta2gamma2s) receptor or human alpha7, alpha4beta2, alpha1beta1deltagamma and alpha1beta1delta epsilon nicotinic acetylcholine receptors expressed in Xenopus oocytes were examined using two-electrode voltage clamp. Tetrandrine inhibited the function of acetylcholine nicotinic receptors, but it had no effect on GABA(A) receptors. Potency of inhibition was influenced by the receptor subtype and the rank order was alpha4beta2>alpha7>alpha1beta1deltagamma congruent with alpha1beta1delta epsilon. Functional inhibition of alpha4beta2 and alpha1beta1deltagamma receptors was noncompetitive, but only inhibition of alpha1beta1deltagamma receptors was voltage-dependent. Binding of 125I-alpha-bungarotoxin to alpha1beta1deltagamma or 3H-cytisine to alpha4beta2 receptors was also inhibited by tetrandrine, but inhibition was noncompetitive and required concentrations higher than those needed to inhibit receptor function. Inhibition of both alpha7 receptor function and binding of 125I-alpha-bungarotoxin to alpha7 receptor were mixed competitive/noncompetitive and occurred at a similar concentration range.     

Study of the calcium dynamics of the human alpha4beta2, alpha3beta4 and alpha1beta1gammadelta nicotinic acetylcholine receptors

In this study three major subtypes of nicotinic acetylcholine receptors were characterized pharmacologically using the calcium influx through the ion channel as a robust functional assay system. Human alpha3beta4 receptors and alpha4beta2 receptors were cloned and stably expressed in HEK293 cells. [(125)I]epibatidine saturation binding yielded a B(max) of 4420+/-840 fmol/mg protein for the alpha4beta2 receptor ( n=4) and 518+/-15 fmol/mg protein for the alpha3beta4 receptor ( n=4). As a source for muscle type of nicotinic receptor, the TE671 cell line was used which expresses endogenously the human fetal alpha1beta1gammadelta subtype of nicotinic receptor. Stimulation of these nicotinic receptor subtypes in the different cell lines led to calcium transients that peaked 5-10 s after agonist application and declined thereafter. Eleven agonists were tested in this study and their efficacy and potency at the three nicotinic receptor subtypes were determined (epibatidine, ABT594, anatoxin, ABT418, nicotine, DMPP, cytisine, ABT089, choline, GTS21, AAR17779). This pharmacological characterization of agonist-induced elevation of intracellular free Ca(2+) revealed a distinct rank order of agonist potency for each receptor subtype. Epibatidine showed at all three subtypes the highest potency and was a full agonist. The agonist-elicited response could be blocked by co-incubation of different antagonists from which mecamylamine did not display a strong subtype specificity. These data illustrate that the assessment of calcium transients upon receptor stimulation is a powerful tool for rapid examination of the functional properties of nicotinic receptors.     

Expression and functional characterisation of a human chimeric nicotinic receptor with alpha6beta4 properties

Despite being cloned several years ago, the expression of functional nicotinic acetylcholine receptors containing the human alpha6 subunit in recombinant mammalian cell lines has yet to be demonstrated. The resulting lack of selective ligands has hindered the evaluation of the role of alpha6-containing nicotinic receptors. We report that functional channels were recorded following co-transfection of human embryonic kidney (HEK-293) cells with a chimeric alpha6/alpha4 subunit and the beta4 nicotinic receptor subunit. They displayed an agonist rank order potency of epibatidinez.>1,1-dimethyl-4-phenylpiperazinium (DMPP)>/=cytisine>acetylcholine>nicotine measured in a fluorescent imaging plate reader assay. Nicotine, cytisine, DMPP and epibatidine displayed partial agonist properties whilst alpha-conotoxin MII and methyllycaconitine blocked the functional responses elicited by acetylcholine stimulation. Co-transfection of the alpha6/alpha4 chimera with the beta2 nicotinic receptor subunit did not result in functional receptors. The human alpha6/alpha4beta4 chimeric nicotinic receptor expressed in HEK-293 cells may provide a valuable tool for the generation of subtype specific ligands.     

Alkaloids indolizidine 235B', quinolizidine 1-epi-207I, and the tricyclic 205B are potent and selective noncompetitive inhibitors of nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors are key molecules in cholinergic transmission in the nervous system. Because of their structural complexity, only a limited number of subtype-specific agonists and antagonists are available to study nicotinic receptor functions. To overcome this limitation, we used voltageclamp recordings to examine the effects of several frog skin alkaloids on acetylcholine-elicited currents in Xenopus laevis oocytes expressing major types of neuronal nicotinic receptors (alpha4beta2, alpha7, alpha3beta2, alpha3beta4, and alpha4beta4). We found that the 5,8-disubstituted indolizidine (-)-235B' acted as a potent noncompetitive blocker of alpha4beta2 nicotinic receptors (IC50 = 74 nM). This effect was highly selective for alpha4beta2 receptors compared with alpha3beta2, alpha3beta4, and alpha4beta4 receptors. The inhibition of alpha4beta2 currents by (-)-235B' was more pronounced as the acetylcholine concentration increased (from 10 nM to 100 microM). Moreover, the blockade of alpha4beta2 currents by (-)-235B' was voltage-dependent (more pronounced at hyperpolarized potentials) and use-dependent, indicating that (-)-235B' behaves as an open-channel blocker of this receptor. Several other 5,8-disubstituted indolizidines (5-n-propyl-8-n-butylindolizidines), two 5,6,8-trisubstituted indolizidines ((-)-223A and (+)-6-epi-223A), and a 1,4-disubstituted quinolizidine ((+)-207I) were less potent than (-)-235B', and none showed selectivity for alpha4beta2 receptors. The quinolizidine (-)-1-epi-207I and the tricyclic (+)-205B had 8.7- and 5.4-fold higher sensitivity, respectively, for inhibition of the alpha7 nicotinic receptor than for inhibition of the alpha4beta2 receptor. These results show that frog alkaloids alter the function of nicotinic receptors in a subtype-selective manner, suggesting that an analysis of these alkaloids may aid in the development of selective drugs to alter nicotinic cholinergic functions.     

Incorporation of the beta3 subunit has a dominant-negative effect on the function of recombinant central-type neuronal nicotinic receptors

The beta3 neuronal nicotinic subunit is localized in dopaminergic areas of the central nervous system, in which many other neuronal nicotinic subunits are expressed. So far, beta3 has only been shown to form functional receptors when expressed together with the alpha3 and beta4 subunits. We have systematically tested in Xenopus laevis oocytes the effects of coexpressing human beta3 with every pairwise functional combination of neuronal nicotinic subunits likely to be relevant to the central nervous system. Expression of alpha7 homomers or alpha/beta pairs (alpha2, alpha3, alpha4, or alpha6 together with beta2 or beta4) produced robust nicotinic currents for all combinations, save alpha6beta2 and alpha6beta4. Coexpression of wild-type beta3 led to a nearly complete loss of function (measured as maximum current response to acetylcholine) for alpha7 and for all functional alpha/beta pairs except for alpha3beta4. This effect was also seen in hippocampal neurons in culture, which lost their robust alpha7-like responses when transfected with beta3. The level of surface expression of nicotinic binding sites (alpha3beta4, alpha4beta2, and alpha7) in tsA201 cells was only marginally affected by beta3 expression. Furthermore, the dominant-negative effect of beta3 was abolished by a valine-serine mutation in the 9' position of the second transmembrane domain of beta3, a mutation believed to facilitate channel gating. Our results show that incorporation of beta3 into neuronal nicotinic receptors other than alpha3beta4 has a powerful dominant-negative effect, probably due to impairment in gating. This raises the possibility of a novel regulatory role for the beta3 subunit on neuronal nicotinic signaling in the central nervous system.     

Diverse actions of neonicotinoids on chicken alpha7, alpha4beta2 and Drosophila-chicken SADbeta2 and ALSbeta2 hybrid nicotinic acetylcholine receptors expressed in Xenopus laevis oocytes

The 2-nitroimino-imidazolidine and related moieties are structural features of neonicotinoid insecticides acting on nicotinic acetylcholine receptors (nicotinic AChRs). To evaluate these moieties in neonicotinoid interactions with nicotinic AChR alpha subunits, the actions of imidacloprid and related compounds on the chicken alpha7, alpha4beta2 and Drosophila melanogaster-chicken hybrid (SADbeta2 and ALSbeta2) receptors expressed in Xenopus laevis oocytes were studied by voltage-clamp electrophysiology. Imidacloprid and nitenpyram were partial agonists and a nitromethylene analog of imidacloprid (CH-IMI) was a full agonist of the alpha7 receptor, whereas their agonist actions on the alpha4beta2 receptor were very weak, contrasting with full agonist actions of DN-IMI, a desnitro derivative of imidacloprid. The neonicotinoids and DN-IMI were either full or partial agonists of the SADbeta2 receptors. Nitenpyram and DN-IMI were partial agonists of the ALSbeta2 receptor, whereas imidacloprid and CH-IMI scarcely activated the ALSbeta2 receptor. Imidacloprid and CH-IMI in fact suppressed ACh-induced responses of the ALSbeta2 receptor, whereas imidacloprid potentiated and CH-IMI suppressed ACh-induced responses of the alpha4beta2 receptor. These results suggest that interactions with alpha subunits of the 2-nitroimino-imidazolidine moiety of imidacloprid play a role in determining not only agonist and antagonist actions on all four receptors, but also the potentiation of ACh-induced responses of the alpha4beta2 receptor.     

Characterization of nicotinic acetylcholine receptor-mediated noradrenaline release from the isolated rat stomach

We characterized nicotinic acetylcholine receptor-mediated noradrenaline release from the isolated, vascularly perfused rat stomach. The stomach was perfused via the coeliac artery with Krebs-Ringer solution at a constant flow rate of 4 ml per minute. Endogenous noradrenaline released into the perfusate was electrochemically measured using high-performance liquid chromatography. Nicotinic receptor agonists were applied once into the perfusion medium for 2 min and nicotinic receptor antagonists were administered throughout the experiments. The (-)-nicotine (3x10(-5) M)-induced noradrenaline release was abolished by tetrodotoxin and hexamethonium and partially blocked by dihydro-beta-erythroidine (up to 10(-5) M) (a relatively selective antagonist of alpha4beta2 nicotinic receptors) and abolished by mecamylamine (10(-5) M) (a relatively selective antagonist of alpha3beta4 nicotinic receptors), but not influenced by alpha-bungarotoxin (3x10(-7) M) or alpha-conotoxin ImI (10(-6) M) (antagonists of alpha7 nicotinic receptors). (+/-)-Epibatidine (3x10(-7) M) (a very potent, but non-selective agonist) and (-)-cytisine (3x10(-4) M) (an agonist of beta4 nicotinic receptors) effectively evoked the release of noradrenaline, while (E)-N-methyl-4-(3-pyridinyl)-3-butene-1-amine (RJR-2403) (up to 10(-4) M) (an agonist of alpha4beta2 nicotinic receptors) had no effect. The potency of these agonists was as followed; (+/-)-epibatidine>(-)-nicotine>(-)-cytisine>RJR -2403. These results are compatible with the published view that alpha3beta4 nicotinic receptors are predominant in other parts of the autonomic nervous system. These receptors (probably located on the gastric sympathetic ganglia) are involved in the release of noradrenaline from the rat stomach.     

C3-halogenation of cytisine generates potent and efficacious nicotinic receptor agonists

Neuronal nicotinic acetylcholine receptors subserve predominantly modulatory roles in the brain, making them attractive therapeutic targets. Natural products provide key leads in the quest for nicotinic receptor subtype-selective compounds. Cytisine, found in Leguminosae spp., binds with high affinity to alpha4beta2* nicotinic receptors. We have compared the effect of C3 and C5 halogenation of cytisine and methylcytisine (MCy) on their interaction with native rat nicotinic receptors. 3-Bromocytisine (3-BrCy) and 3-iodocytisine (3-ICy) exhibited increased binding affinity (especially at alpha7 nicotinic receptors; Ki approximately 0.1 microM) and functional potency, whereas C5-halogenation was detrimental. 3-BrCy and 3-ICy were more potent than cytisine at evoking [3H]dopamine release from striatal slices (EC50 approximately 11 nM), [3H]noradrenaline release from hippocampal slices (EC50 approximately 250 nM), increases in intracellular Ca2+ in PC12 cells and inward currents in Xenopus oocytes expressing human alpha3beta4 nicotinic receptor (EC50 approximately 2 microM). These compounds were also more efficacious than cytisine. C3-halogenation of cytisine is proposed to stabilize the open conformation of the nicotinic receptor but does not enhance subtype selectivity.     

Conformational mobility of immobilized proteins

Cellular membrane fragments have been immobilized on the surface of a silica-based liquid chromatographic support and on the surface of glass capillaries to create immobilized receptor and drug transporter columns. These columns have included phases containing one subtype of the nicotinic receptor (alpha3beta2, alpha3beta4, alpha4beta2, alpha4beta4) and the P-glycoprotein transporter. A key question in the application of these columns to drug discovery and development is the ability of the immobilized receptor or transporter to undergo ligand and/or co-factor induced conformational changes. Using frontal affinity chromatographic techniques and non-linear chromatographic techniques it has been demonstrated that the immobilized nicotinic receptors undergo agonist-induced conformational shifts from the resting to desensitized states with corresponding changes in binding affinities and enantioselectivities. Ligand-induced allosteric interactions and ATP-driven conformational changes have also been demonstrated with the immobilized Pgp stationary phase. The results demonstrate that the immobilized proteins retained their ability to undergo conformational mobility and that this is an attractive alternative to allow for the full characterization of multiple protein conformations.     

beta3 subunit is present in different nicotinic receptor subtypes in chick retina

Although the neuronal nicotinic beta3 subunit was cloned several years ago, it has only recently been shown to form heteromeric channels when associated with other nicotinic subunits, and very little information is available concerning its assembly in the native nicotinic receptors of the nervous system. Using subunit-specific antibodies and immunoprecipitation experiments, we have identified the retina as being the chick central nervous system (CNS) area that expresses the highest level of the beta3 subunit. Sequential immunopurification experiments showed that there are at least two populations of beta3-containing receptors in chick retina: in one, the beta3 subunit is associated with the alpha6 and beta4 subunits; in the other more heterogeneous population, the beta3 subunit is associated with the alpha2, alpha3, alpha4, beta2 and beta4 subunits. Both of these receptor populations bind [3H]epibatidine and a number of nicotinic receptor agonists with high affinity (nM) and nicotinic receptor antagonists with a lower affinity (microM). The greatest pharmacological difference between the two populations is the affinity for the alpha-conotoxin MII, which inhibits binding to alpha6-containing receptors and not that to beta3-containing receptors. We also searched for the presence of the beta3 subunit associated with the alpha-bungarotoxin binding subunits alpha7 and/or alpha8 in retina and chick brain. Immunoprecipitation studies using anti-beta3 antibodies did not detect any specific alpha-bungarotoxin labeled receptors, thus, indicating that the beta3 subunit is not present in the alpha-bungarotoxin receptors of these areas.     

Inefficient cell-surface expression of hybrid complexes formed by the co-assembly of neuronal nicotinic acetylcholine receptor and serotonin receptor subunits.

Previous studies have demonstrated that relatively low levels of alpha4beta2 neuronal nicotinic acetylcholine receptors (nAChRs) are expressed on the cell surface of transfected mammalian cell lines but that surface expression levels can be dramatically up-regulated by co-expression of these subunits with chimeric subunits containing the N-terminal portion of the neuronal nAChR alpha4 or beta2 subunits together with the C-terminal domain of the 5-HT(3A) subunit. Recent work has also suggested that the nAChR alpha4 subunit can co-assemble in a "promiscuous" manner with the serotonin receptor 5-HT(3A) subunit to form functional hybrid receptors. In this study we have examined whether co-assembly of either alpha4 or beta2 with 5-HT(3A) itself (rather than with the alpha4/5-HT(3A) or beta2/5-HT(3A) subunit chimeras) can also facilitate cell surface expression of alpha4 and beta2 subunits in transfected mammalian cells. Evidence has been obtained by immunoprecipitation, cell-surface antibody binding and radioligand binding which indicates that the 5-HT(3A) can co-assemble with both the alpha4 and beta2 nAChR subunits. We conclude, however, that co-assembly of 5-HT(3A) with either alpha4 or beta2 does not result in efficient cell surface expression of the nAChR subunits and that co-assembled hybrid (nAChR subunit + 5-HT(3)R subunit) receptor complexes are largely retained within the cell.