Resistance to Inhibitors of Cholinesterase 3 (Ric-3) Expression Promotes Selective Protein Associations with the Human ?7-Nicotinic Acetylcholine Receptor Interactome.
ABSTRACT: The ?7-nicotinic acetylcholine receptor (?7-nAChR) is a ligand-gated ion channel widely expressed in vertebrates and is associated with numerous physiological functions. As transmembrane ion channels, ?7-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 ?7-nAChRs. The putative chaperone resistance to inhibitors of cholinesterase 3 (Ric-3) has been reported to interact with, and enhance the surface expression of, ?7-nAChRs. In this study, we identified proteins that associate with ?7-nAChRs when Ric-3 is expressed. Using ?-bungarotoxin (?-bgtx), we isolated and compared ?7-nAChR-associated proteins from two stably transfected, human tumor-derived cell lines: SH-EP1-h?7 expressing human ?7-nAChRs and the same cell line further transfected to express Ric-3, SH-EP1-h?7-Ric-3. Mass spectrometric analysis of peptides identified thirty-nine proteins that are associated with ?7-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 ?-bgtx may be used to isolate and identify ?7-nAChRs as well as how the expression of chaperones such as Ric-3 can influence proteins associating with ?7-nAChRs. These associating proteins may alter activities of ?7-nAChRs to expand their functionally-relevant repertoire as well as to affect biogenesis and membrane trafficking of ?7-nAChRs.
Project description:The ?7-nicotinic acetylcholine receptor (?7-nAChR) is a ligand-gated ion channel that is expressed widely in vertebrates and is the principal high-affinity ?-bungarotoxin (?-bgtx) binding protein in the mammalian CNS. ?7-nAChRs associate with proteins that can modulate its properties. The ?7-nAChR interactome is the summation of proteins interacting or associating with ?7-nAChRs in a protein complex. To identify an ?7-nAChR interactome in neural tissue, we isolated ?-bgtx-affinity protein complexes from wild-type and ?7-nAChR knockout (?7 KO) mouse whole brain tissue homogenates using ?-bgtx-affinity beads. Affinity precipitated proteins were trypsinized and analyzed with an Orbitrap Fusion mass spectrometer. Proteins isolated with the ?7-nAChR specific ligand, ?-bgtx, were determined to be ?7-nAChR associated proteins. The ?7-nAChR subunit and 120 additional proteins were identified. Additionally, 369 proteins were identified as binding to ?-bgtx in the absence of ?7-nAChR expression, thereby identifying nonspecific proteins for ?7-nAChR investigations using ?-bgtx enrichment. These results expand on our previous investigations of ?7-nAChR interacting proteins using ?-bgtx-affinity bead isolation by controlling for differences between ?7-nAChR and ?-bgtx-specific proteins, developing an improved protein isolation methodology, and incorporating the latest technology in mass spectrometry. The ?7-nAChR interactome identified in this study includes proteins associated with the expression, localization, function, or modulation of ?7-nAChRs, and it provides a foundation for future studies to elucidate how these interactions contribute to human disease.
Project description:Alpha6-containing nicotinic acetylcholine receptors are primarily found in neurons of the midbrain dopaminergic (DA) system, suggesting these receptors are potentially involved in drug reward and dependence. Here, we report a novel effect that cocaine directly inhibits ?6N/?3C?2?3-nAChR (?6*-nAChRs) function. Human ?6*-nAChRs were heterologously expressed within cells of the SH-EP1 cell line for functional characterization. Mechanically dissociated DA neurons from mouse ventral tegmental area (VTA) were used as a model of presynaptic ?6*-nAChR activation since this method preserves terminal boutons. Patch-clamp recordings in whole-cell configuration were used to measure ?6*-nAChR function as well as evaluate the effects of cocaine. In SH-EP1 cells containing heterologously expressed human ?6*-nAChRs, cocaine inhibits nicotine-induced inward currents in a concentration-dependent manner with an IC50 value of 30 ?M. Interestingly, in the presence of 30 ?M cocaine, the maximal current response of the nicotine concentration-response curve is reduced without changing nicotine's EC50 value, suggesting a noncompetitive mechanism. Furthermore, analysis of whole-cell current kinetics demonstrated that cocaine slows nAChR channel activation but accelerates whole-cell current decay time. Our findings demonstrate that cocaine-induced inhibition occurs solely with bath application, but not during intracellular administration, and this inhibition is not use-dependent. Additionally, in Xenopus oocytes, cocaine inhibits both ?6N/?3C?2?3-nAChRs and ?6M211L/?3IC?2?3-nCAhRs similarly, suggesting that cocaine may not act on the ?3 transmembrane domain of chimeric ?6N/?3C?2?3-nAChR. In mechanically isolated VTA DA neurons, cocaine abolishes ?6*-nAChR-mediated enhancement of spontaneous inhibitory postsynaptic currents (sIPSCs). Collectively, these studies provide the first evidence that cocaine directly inhibits the function of both heterologously and naturally expressed ?6*-nAChRs. These findings suggest that ?6*-nAChRs may provide a novel pharmacological target mediating the effects of cocaine and may underlie a novel mechanism of cocaine reward and dependence.
Project description:Nothing is known about the regulation of nicotinic acetylcholine receptors (nAChRs) in hair cells of the inner ear. MuSK, rapsyn and RIC-3 are accessory molecules associated with muscle and brain nAChR function. We demonstrate that these accessory molecules are expressed in the inner ear raising the possibility of a muscle-like mechanism for clustering and assembly of nAChRs in hair cells. We focused our investigations on rapsyn and RIC-3. Rapsyn interacts with the cytoplasmic loop of nAChR alpha9 subunits but not nAChR alpha10 subunits. Although rapsyn and RIC-3 increase nAChR alpha9 expression, rapsyn plays a greater role in receptor clustering while RIC-3 is important for acetylcholine-induced calcium responses. Our data suggest that RIC-3 facilitates receptor function, while rapsyn enhances receptor clustering at the cell surface.
Project description: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, ?3?4-nAChR expressed in native SH-SY5Y cells. ?3?4-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??M. Kinetic analysis showed that cocaine accelerated ?3?4-nAChR desensitization, which caused a reduction of the amplitude of nicotine-induced currents. Co-application of nicotine and cocaine (1.5??M) 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-?S (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??M) was unable to inhibit the nicotine-induced inward current in heterologous expressed ?6/?3?2?3-nAChRs and ?4?2-nAChRs expressed in human SH-EP1 cells. Collectively, our results suggest that cocaine is a potent blocker for native ?3?4-nAChRs expressed in SH-SY5Y cells.
Project description:Neuronal nicotinic acetylcholine receptors containing ?6 subunits (?6*-nAChRs) show highly restricted distribution in midbrain neurons associated with pleasure, reward, and mood control, suggesting an important impact of ?6*-nAChRs in modulating mesolimbic functions. However, the function and pharmacology of ?6*-nAChRs remain poorly understood because of the lack of selective agonists for ?6*-nAChRs and the challenging heterologous expression of functional ?6*-nAChRs in mammalian cell lines. In particular, the ?6 subunit is commonly co-expressed with ?4*-nAChRs in the midbrain, which masks ?6*-nAChR (without ?4) function and pharmacology. In this study, we systematically profiled the pharmacology and function of ?6*-nAChRs and compared these properties with those of ?4?2 nAChRs expressed in the same cell line. Heterologously expressed human ?6/?3 chimeric subunits (?6 N-terminal domain joined with ?3 trans-membrane domains and intracellular loops) with ?2 and ?3 subunits in the human SH-EP1 cell line (?6*-nAChRs) were used. Patch-clamp whole-cell recordings were performed to measure these receptor-mediated currents. Functionally, the heterologously expressed ?6*-nAChRs exhibited excellent function and showed distinct nicotine-induced current responses, such as kinetics, inward rectification and recovery from desensitization, compared with ?4?2-nAChRs. Pharmacologically, ?6*-nAChR was highly sensitive to the ?6 subunit-selective antagonist ?-conotoxin MII but had lower sensitivity to mecamylamine and dihydro-?-erythroidine. Nicotine and acetylcholine were found to be full agonists for ?6*-nAChRs, whereas epibatidine and cytisine were determined to be partial agonists. Heterologously expressed ?6*-nAChRs exhibited pharmacology and function distinct from those of ?4?2-nAChRs, suggesting that ?6*-nAChRs may mediate different cholinergic signals. Our ?6*-nAChR expression system can be used as an excellent cell model for future investigations of ?6*-nAChR function and pharmacology.
Project description:<h4>Background and purpose</h4>Expression of ?7 nicotinic acetylcholine receptors (nAChRs) and their role in exocytosis have not yet been examined in human chromaffin cells.<h4>Experimental approach</h4>To characterize these receptors and investigate their function, patch-clamp experiments were performed in human chromaffin cells from organ donors.<h4>Key results</h4>The nicotinic current provoked by 300µM ACh in voltage-clamped cells was blocked by the nicotinic receptor antagonists ?-bungarotoxin (?-Bgtx; 1µM; 6 ± 1.7%) or methyllycaconitine (MLA; 10nM; 7 ± 1.6%), respectively, in an irreversible and reversible manner, without affecting exocytosis. Choline (10mM) pulses induced a biphasic current with an initial quickly activated (5.5 ± 0.4ms rise time) and inactivated component (8.5 ± 0.4ms time constant) (termed ?7), which was blocked by ?-Bgtx or MLA, followed by a slower component (non-?7). ?7 nAChR currents were dissected by blocking the non-?7 nAChR current component of the ACh and choline response with the ?6* nAChR blocker ?-conotoxin (?-Ctx) MII[S4A, E11A, L15A]. PNU-282987, an ?7 nAChR-specific agonist, elicited rapidly activated and rapidly inactivated currents. ?7 nAChR-positive allosteric modulators, such as 5-hydroxyindole (1mM) and PNU-120596 (10µM), potentiated responses that were blocked by ?-Bgtx or MLA. Exocytosis was evoked by depolarization-elicited ?7 nAChR currents, using choline in the presence of ?-Ctx MII[MS4A, E11A, L15A] or PNU-282987 as agonists.<h4>Conclusions and implications</h4>Our electrophysiological recordings of pure ?7 nAChR currents elicited by rapid application of agonists demonstrated that functional ?7 nAChRs are expressed and contribute to depolarization-elicited exocytosis in human chromaffin cells.
Project description:Several nicotinic acetylcholine receptor (nAChR) subunits have been engineered as fluorescent protein (FP) fusions and exploited to illuminate features of nAChRs. The aim of this work was to create a FP fusion in the nAChR alpha7 subunit without compromising formation of functional receptors.A gene construct was generated to introduce yellow fluorescent protein (YFP), in frame, into the otherwise unaltered, large, second cytoplasmic loop between the third and fourth transmembrane domains of the mouse nAChR alpha7 subunit (alpha7Y). SH-EP1 cells were transfected with mouse nAChR wild type alpha7 subunits (alpha7) or with alpha7Y subunits, alone or with the chaperone protein, hRIC-3. Receptor function was assessed using whole-cell current recording. Receptor expression was measured with (125)I-labeled alpha-bungarotoxin (I-Bgt) binding, laser scanning confocal microscopy, and total internal reflectance fluorescence (TIRF) microscopy.Whole-cell currents revealed that alpha7Y nAChRs and alpha7 nAChRs were functional with comparable EC(50) values for the alpha7 nAChR-selective agonist, choline, and IC(50) values for the alpha7 nAChR-selective antagonist, methyllycaconitine. I-Bgt binding was detected only after co-expression with hRIC-3. Confocal microscopy revealed that alpha7Y had primarily intracellular rather than surface expression. TIRF microscopy confirmed that little alpha7Y localized to the plasma membrane, typical of alpha7 nAChRs.nAChRs composed as homooligomers of alpha7Y subunits containing cytoplasmic loop YFP have functional, ligand binding, and trafficking characteristics similar to those of alpha7 nAChRs. alpha7Y nAChRs may be used to elucidate properties of alpha7 nAChRs and to identify and develop novel probes for these receptors, perhaps in high-throughput fashion.
Project description:RIC-3 is a transmembrane protein which acts as a molecular chaperone of nicotinic acetylcholine receptors (nAChRs). For some nAChR subtypes (such as homomeric alpha7 neuronal nAChRs), RIC-3 is required for efficient receptor folding, assembly and functional expression. In contrast, for other nAChR subtypes (such as heteromeric alpha4beta2 neuronal nAChRs) there have been reports that RIC-3 can both enhance and reduce levels of functional expression. There is also evidence that RIC-3 can modulate maturation of the closely related 5-hydroxytryptamine (5-HT) receptor (5-HT(3)R). As with heteromeric nAChRs, apparently contradictory results have been reported for the influence of RIC-3 on 5-HT(3)R maturation in different expression systems. Recent evidence indicates that these differences in RIC-3 chaperone activity may be influenced by the host cell, suggesting that other proteins may play an important role in modulating the effects of RIC-3 as a chaperone. RIC-3 was originally identified in the nematode Caenorhabditis elegans as the protein encoded by the gene ric-3 (resistance to inhibitors of cholinesterase) and has subsequently been cloned and characterized from mammalian and insect species. This review provides a brief history of RIC-3; from the identification of the ric-3 gene in C. elegans in 1995 to the more recent demonstration of its activity as a nAChR chaperone.
Project description: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 ?6*-nicotinic acetylcholine receptor (?6*-nAChR) function when heterologously expressed in SH-EP1 human epithelial cells, we found that 0.1-5 mM EtOH significantly enhances ?6*-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 ?6*-nAChRs since it did not affect ?3?4-, ?4?2-, or ?7-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 ?6*-nAChR antagonist, ?-conotoxin MII, suggesting a role for native ?6*-nAChRs in low-dose EtOH effects. Collectively, our data suggest that ?6*-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.
Project description:We investigated assembly and function of nicotinic acetylcholine receptors (nAChRs) composed of ?7 and ?2 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 ?7 and ?2 subunits colocalize. Förster resonance energy transfer between fluorescently tagged subunits strongly suggested that ?7 and ?2 subunits coassemble. Total internal reflection fluorescence microscopy revealed that assemblies localized to filopodia-like processes of SH-EP1 cells. Gain-of-function ?7 and ?2 subunits confirmed that these subunits coassemble within functional receptors. Moreover, ?7?2 nAChRs composed of wild-type subunits or fluorescently tagged subunits had pharmacological properties similar to those of ?7 nAChRs, although amplitudes of ?7?2 nAChR-mediated, agonist-evoked currents were generally ~2-fold lower than those for ?7 nAChRs. It is noteworthy that ?7?2 nAChRs displayed sensitivity to low concentrations of the antagonist dihydro-?-erythroidine that was not observed for ?7 nAChRs at comparable concentrations. In addition, cysteine mutants revealed that the ?7-?2 subunit interface does not bind ligand in a functionally productive manner, partly explaining lower ?7?2 nAChR current amplitudes and challenges in identifying the function of native ?7?2 nAChRs. On the basis of our findings, we have constructed a model predicting receptor function that is based on stoichiometry and position of ?2 subunits within the ?7?2 nAChRs.