Nicotinic acetylcholine receptor subunits and receptor activity in the epithelial cell line HT29

In the present study we have used RT-PCR to investigate nicotinic acetylcholine receptor (nAChR) subunit expression, and studied the effect of nicotine on TNFalpha-induced cytokine (IL-8) release in the epithelial cell line HT29. RNA was extracted using a commercial kit and amplified by RT-PCR. RT-PCR products were separated by electrophoresis and visualised using ethidium bromide. IL-8 release was measured by ELISA from cells activated for 6 h with TNFalpha (50 ng ml(-1)) in the absence and presence of nicotine (10(-11)-10(-6) M). HT29 cells contained mRNA for beta1, alpha4, alpha5, and alpha7 nAChR subunits. Activation of HT29 cells increased IL-8 release from undetectable amounts to 3.92 +/- 0.51 ng ml(-1) (n = 5). Nicotine significantly inhibited TNFalpha-induced IL-8 release in a concentration related manner with peak inhibition occurring at 10(-7) M (2.39 +/- 0.78 ng ml(-1), n = 5). Our data suggests that, while HT29 cells express mRNA for nAChR subunits, the only nAChR subunits that could form functional receptors and inhibit IL-8 release are alpha7.     

Evolution of nicotinic acetylcholine receptor subunits

A phylogenetic tree of a gene family of nicotinic acetylcholine receptorsubunits was constructed using 84 nucleotide sequences of receptor subunitsfrom 18 different species in order to elucidate the evolutionary origin ofreceptor subunits. The tree constructed showed that the common ancestor ofall subunits may have appeared first in the nervous system. Moreover, wesuggest that the alpha 1 subunits in the muscle system originated from thecommon ancestor of alpha 2, alpha 3, alpha 4, alpha 5, alpha 6, and beta 3in the nervous system, whereas the beta 1, gamma, delta, and epsilonsubunits in the muscle system shared a common ancestor with the beta 2 andbeta 4 subunits in the nervous system. Using the ratio (f) of the number ofnonsynonymous substitutions to that of synonymous substitutions, wepredicted the functional importance of subunits. We found that the alpha 1and alpha 7 subunits had the lowest f values in the muscle and nervoussystems, respectively, indicating that very strong functional constraintswork on these subunits. This is consistent with the fact that the alpha 1subunit has sites binding to the ligand, and the alpha 7-containingreceptor regulates the release of the transmitter. Moreover, the windowanalysis of the f values showed that strong functional constraints work onthe so-called M2 region in all five types of muscle subunits. Thus, thewindow analysis of the f values is useful for evaluating the degree offunctional constraints in not only the entire gene region, but also thewithin-gene subregion.     

Nicotinic acetylcholine receptors assembled from the alpha7 and beta3 subunits.

Intracellular recordings were performed in voltage-clamped Xenopus oocytes upon injection with a mixture of cDNAs encoding the beta3 and mutant alpha7 (L247Talpha7) neuronal nicotinic acetylcholine receptor (nAChR) subunits. The expressed receptors maintained sensitivity to methyllycaconitine and to alpha-bungarotoxin but exhibited a functional profile strikingly different from that of the homomeric L247Talpha7 receptor. The heteromeric L247Talpha7beta3 nAChR had a lower apparent affinity and a faster rate of desensitization than L247Talpha7 nAChR, exhibited nonlinearity in the I-V relationship, and was inhibited by 5-hydroxytryptamine, much like wild type alpha7 (WTalpha7) nAChR. Single channel recordings in cell-attached mode revealed unitary events with a slope conductance of 19 picosiemens and a lifetime of 5 ms, both values being much smaller than those of the homomeric receptor channel. Upon injection with a mixture of WTalpha7 and beta3 cDNAs, clear evidence was obtained for the plasma membrane assembly of heteromeric nAChRs, although ACh could not activate these receptors. It is concluded that beta3, long believed to be an orphan subunit, readily co-assembles with other subunits to form heteromeric receptors, some of which may be negative regulators of cholinergic function.     

Nicotinic acetylcholine receptor genes on chromosome 15q25.1 are associated with nicotine and opioid dependence severity

A locus on chromosome 15q25.1 previously implicated in nicotine, alcohol, and cocaine dependence, smoking, and lung cancer encodes subunits of the nicotinic acetylcholine receptor (nAChR) expressed in the mesolimbic system and thought to mediate substance dependence. Opioid dependence severity (ODS), nicotine dependence severity (NDS), smoking status and quantity, and the number of attempts to quit were assessed using questionnaire instruments in 505 subjects who were prescribed opioid medications for chronic pain in outpatient practice sites. Multivariate regression was used to test for genetic association of these phenotypes with 5 SNPs in the nAChR gene cluster on chromosome 15q25.1, adjusting for background variables. A coding variant in CHRNA5 (rs16969968[A]) was significantly associated with 1.4-unit higher ODS (p < 0.00017). A variant in the 3′ untranslated region of CHRNA3 (rs660652[G]) was significantly associated with 1.7-fold higher odds of lifetime smoking (p < 0.0092), 1.1-unit higher NDS (p < 0.0007), 0.7 more pack-years of cigarette smoking (p < 0.0038), and 0.8 more lifetime attempts to quit (p < 0.0084). Our data suggest an association of DNA variants in the nAChR gene cluster on chromosome 15q25.1 with ODS, as well as NDS and related smoking phenotypes. While the association of this locus with NDS and smoking phenotypes is well known, the association with ODS, a dimension of opioid substance dependence, is novel and requires verification in independent studies.     

Nicotinic acetylcholine receptors of Drosophila: three subunits encoded by genomically linked genes can co-assemble into the same receptor complex

The second beta-like subunit (SBD) is a putative structural subunit of Drosophila melanogaster nicotinic acetylcholine receptors (nAChRs). Here we have produced specific antibodies against SBD to study, which other nAChR subunits can co-assemble with SBD in receptor complexes of the Drosophila nervous system. Immunohistochemical studies in the adult optic lobe revealed that SBD has a distribution similar to that of the alpha-subunit ALS in the synaptic neuropil. The subunits ALS, D(alpha)2 and SBD can be co-purified by alpha-bungarotoxin affinity chromatography. Moreover, anti-SBD antibodies co-precipitate ALS and D(alpha)2 and, vice versa, ALS and D(alpha)2 antibodies co-immunoprecipitate SBD protein. Two-step immunoaffinity chromatography with immobilized antibodies against ALS and D(alpha)2 revealed the existence of nAChR complexes that include ALS, D(alpha)2 and SBD as integral components. Interestingly, the genes encoding these three subunits appear to be directly linked in the Drosophila genome at region 96 A of the third chromosome. In addition, SBD appears to be a component of a different receptor complex, which includes the ARD protein as an additional beta-subunit, but neither ALS nor D(alpha)2 nor the third alpha-subunit D(alpha)3. These findings suggest a considerable complexity of the Drosophila nicotinic receptor system.     

Rapsyn-mediated clustering of acetylcholine receptor subunits requires the major cytoplasmic loop of the receptor subunits

During synaptogenesis at the neuromuscular junction, nicotinic acetylcholine receptors (AChRs) are organized into high-density postsynaptic clusters that are critical for efficient synaptic transmission. Rapsyn, an AChR associated cytoplasmic protein, is essential for the aggregation and immobilization of AChRs at the neuromuscular junction. Previous studies have shown that when expressed in nonmuscle cells, both assembled and unassembled AChR subunits are clustered by rapsyn, and the clustering of the alpha subunit is dependent on its major cytoplasmic loop. In the present study, we investigated the mechanism of rapsyn-induced clustering of the AChR beta, gamma, and delta subunits by testing mutant subunits for the ability to cocluster with rapsyn in transfected QT6 cells. For each subunit, deletion of the major cytoplasmic loop, between the third and fourth transmembrane domains, dramatically reduced coclustering with rapsyn. Furthermore, each major cytoplasmic loop was sufficient to mediate clustering of an unrelated transmembrane protein. The AChR subunit mutants lacking the major cytoplasmic loops could assemble into alphadelta dimers, but these were poorly clustered by rapsyn unless at least one mutant was replaced with its wild-type counterpart. These results demonstrate that the major cytoplasmic loop of each AChR subunit is both necessary and sufficient for mediating efficient clustering by rapsyn, and that only one such domain is required for rapsyn-mediated clustering of an assembly intermediate, the alphadelta dimer.     

Nicotinic acetylcholine receptors in health and disease

Nicotinic acetylcholine receptors (AChRs) are a family of acetylcholine-gated cation channels that form the predominant excitatory neurotransmitter receptors on muscles and nerves in the peripheral nervous system. AChRs are also expressed on neurons in lower amounts throughout the central nervous system. AChRs are even being reported on unexpected cell types such as keratinocytes. Structures of these AChRs are being determined with increasing precision, but functions of some orphan subunits are just beginning to be established. Functional roles for postsynaptic AChRs in muscle are well known, but in neurons the post-, peri-, extra-, and presynaptic roles of AChRs are just being revealed. Pathogenic roles of AChRs are being discovered in many diseases involving mechanisms ranging from mutations, to autoimmune responses, to the unknown; involving cell types ranging from muscles, to neurons, to keratinocytes; and involving signs and symptoms ranging from muscle weakness to epilepsy, to neurodegenerative disease, to psychiatric disease, to nicotine addiction. Awareness of AChR involvement in some of these diseases has provoked new interests in development of therapeutic agonists for specific AChR subtypes and the use of expressed cloned AChR subunits as possible immunotherapeutic agents. Highlights of recent developments in these areas will be briefly reviewed.     

Nicotinic acetylcholine receptor channel electrostatics determined by diffusion-enhanced luminescence energy transfer

The electrostatic potentials within the pore of the nicotinic acetylcholine receptor (nAChR) were determined using lanthanide-based diffusion-enhanced fluorescence energy transfer experiments. Freely diffusing Tb3+ -chelates of varying charge constituted a set of energy transfer donors to the acceptor, crystal violet, a noncompetitive antagonist of the nAChR. Energy transfer from a neutral Tb3+ -chelate to nAChR-bound crystal violet was reduced 95% relative to the energy transfer to free crystal violet. This result indicated that crystal violet was strongly shielded from solvent when bound to the nAChR. Comparison of energy transfer from positively and negatively charged chelates indicate negative electrostatic potentials of -25 mV in the channel, measured in low ionic strength, and -10 mV measured in physiological ionic strength. Debye-Hückel analyses of potentials determined at various ionic strengths were consistent with 1-2 negative charges within 8 A of the crystal violet binding site. To complement the energy transfer experiments, the influence of pH and ionic strength on the binding of [3H]phencyclidine were determined. The ionic strength dependence of binding affinity was consistent with -3.3 charges within 8 A of the binding site, according to Debye-Hückel analysis. The pH dependence of binding had an apparent pKa of 7.2, a value indicative of a potential near -170 mV if the titratable residues are constituted of aspartates and glutamates. It is concluded that long-range potentials are small and likely contribute little to selectivity or conductance whereas close interactions are more likely to contribute to electrostatic stabilization of ions and binding of noncompetitive antagonists within the channel.     

Relative localization of the bound acetylcholine receptor subunits and neurotoxin

A series of neurotoxin II (Naja naja oxiana) derivatives, each containing one p-azido-[14C]benzoyl group, have been prepared. Those labeled at Leu1, Lys15, Lys25, Lys26, or Lys46 associate specifically with the acetylcholine receptor from the Torpedo marmorata electric organs and form the crosslinks with it as a result of irradiation. Electrophoresis in polyacrylamide gel and gel chromatography revealed the contacts between the neurotoxins and alpha, beta, gamma and delta subunits of the receptor, modification of a particular subunit being governed by the photoactivable group position in the neurotoxin molecule. The differences of the two neurotoxin binding sites in the receptor were demonstrated by analysis of the photoinduced crosslinks under the conditions of one site being blocked by hexa (trifluoroacetyl) neurotoxin II. The mutual arrangement of the two bound neurotoxin molecules was established. On the basis of data obtained, two models for the acetylcholine receptor subunit topography were proposed.     

Characterization of acetylcholine receptor subunits in developing and in denervated mammalian muscle

We have used subunit-specific antibodies to identify and to characterize partially the alpha, beta, gamma, and delta subunits of rat skeletal muscle acetylcholine receptor (AChR) on immunoblots. The alpha subunit of rat muscle is a single band of 42 kDa, whereas the beta subunit has an apparent molecular mass of 48 kDa. Both alpha and beta subunits are glycosylated and contain one or more N-linked oligosaccharide chains that are sensitive to endoglycosidase H digestion. The gamma and delta subunits, on the other hand, each appear as doublets on immunoblots, with apparent molecular masses of 52 kDa (gamma), 48 kDa (gamma') and 58 kDa (delta), 53 kDa (delta'), respectively. In each case, the two bands are structurally related and the lower band is probably the partial degradation product of the corresponding upper band. Each of the four gamma and delta polypeptides is N-glycosylated and contains both endoglycosidase H-sensitive and endoglycosidase H-resistant oligosaccharides. When the AChRs purified from embryonic, neonatal, adult, and denervated adult rat muscles were compared, no differences in the mobilities of alpha, beta, or delta subunits on sodium dodecyl sulfate gels were detected among them, either with or without endoglycosidase treatment. The gamma subunits, which were present in AChRs purified from neonatal, embryonic, or denervated rat muscles, were also identical; no gamma subunit was detected, however, in AChRs of normal adult rat muscle.     

Nicotinic acetylcholine receptor induces lateral segregation of phosphatidic acid and phosphatidylcholine in reconstituted membranes

Purified nicotinic acetylcholine receptor (AChR) protein was reconstituted into synthetic lipid membranes having known effects on receptor function in the presence and absence of cholesterol (Chol). The phase behavior of a lipid system (DPPC/DOPC) possessing a known lipid phase profile and favoring nonfunctional, desensitized AChR was compared with that of a lipid system (POPA/POPC) containing the anionic phospholipid phosphatidic acid (PA), which stabilizes the functional resting form of the AChR. Fluorescence quenching of diphenylhexatriene (DPH) extrinsic fluorescence and AChR intrinsic fluorescence by a nitroxide spin-labeled phospholipid showed that the AChR diminishes the degree of DPH quenching and promotes DPPC lateral segregation into an ordered lipid domain, an effect that was potentiated by Chol. Fluorescence anisotropy of the probe DPH increased in the presence of AChR or Chol and also made apparent shifts to higher values in the transition temperature of the lipid system in the presence of Chol and/or AChR. The values were highest when both Chol and AChR were present, further reinforcing the view that their effect on lipid segregation is additive. These results can be accounted for by the increase in the size of quencher-free, ordered lipid domains induced by AChR and/or Chol. Pyrene phosphatidylcholine (PyPC) excimer (E) formation was strongly reduced owing to the restricted diffusion of the probe induced by the AChR protein. The analysis of Forster energy transfer (FRET) from the protein to DPH further indicates that AChR partitions preferentially into these ordered lipid microdomains, enriched in saturated lipid (DPPC or POPA), which segregate from liquid phase-enriched DOPC or POPC domains. Taken together, the results suggest that the AChR organizes its immediate microenvironment in the form of microdomains with higher lateral packing density and rigidity. The relative size of such microdomains depends not only on the phospholipid polar headgroup and fatty acyl chain saturation but also on AChR protein-lipid interactions. Additional evidence suggests a possible competition between Chol and POPA for the same binding sites on the AChR protein.     

Identification of calcium-binding proteins associated with the human sperm plasma membrane

Background  

The precise composition of the human sperm plasma membrane, the molecular interactions that define domain specific functions, and the regulation of membrane associated proteins during the capacitation process, still remain to be fully understood. Here, we investigated the repertoire of calcium-regulated proteins associated with the human sperm plasma membrane.     

Nicotinic acetylcholine receptor is internalized via a Rac-dependent, dynamin-independent endocytic pathway

Endocytosis of the nicotinic acetylcholine receptor (AChR) is a proposed major mechanism of neuromodulation at neuromuscular junctions and in the pathology of synapses in the central nervous system. We show that binding of the competitive antagonist alpha-bungarotoxin (alphaBTX) or antibody-mediated cross-linking induces the internalization of cell surface AChR to late endosomes when expressed heterologously in Chinese hamster ovary cells or endogenously in C2C12 myocytes. Internalization occurs via sequestration of AChR-alphaBTX complexes in narrow, tubular, surface-connected compartments, which are indicated by differential surface accessibility of fluorescently tagged alphaBTX-AChR complexes to small and large molecules and real-time total internal reflection fluorescence imaging. Internalization occurs in the absence of clathrin, caveolin, or dynamin but requires actin polymerization. alphaBTX binding triggers c-Src phosphorylation and subsequently activates the Rho guanosine triphosphatase Rac1. Consequently, inhibition of c-Src kinase activity, Rac1 activity, or actin polymerization inhibits internalization via this unusual endocytic mechanism. This pathway may regulate AChR levels at ligand-gated synapses and in pathological conditions such as the autoimmune disease myasthenia gravis.     

T cell hybridomas reactive with the acetylcholine receptor and its subunits

A panel of thirty cloned rat-mouse T cell hybridomas was prepared by fusion of acetylcholine receptor (AChR)-reactive rat T cells with the mouse thymoma BW5147. The T cell hybrids were demonstrated to be AChR reactive by their ability to secrete IL 2 in response to either AChR itself or by purified AChR subunits (alpha,beta,gamma, or delta). Various patterns of AChR subunit reactivity were observed, suggesting a predominant recognition of the alpha subunit, and also a considerable cross-reactivity from one subunit to another.     

Functional divergence analysis of vertebrate neuronal nicotinic acetylcholine receptor subunits

Nicotinic acetylcholine receptors (nAChRs) are pentamers formed by subunits from a large multigene family and are highly variable in kinetic, electrophysiological and pharmacological properties. Due to the essential roles of nAChRs in many physiological procedures and diversity in function, identifying the function-related sites specific to each subunit is not only necessary to understand the properties of the receptors but also useful to design potential therapeutic compounds that target these macromolecules for treating a series of central neuronal disorders. By conducting a detailed function divergence analysis on nine neuronal nAChR subunits from representative vertebrate species, we revealed the existence of significant functional variation between most subunit pairs. Specifically, 44 unique residues were identified for the α7 subunit, while another 22 residues that were likely responsible for the specific features of other subunits were detected. By mapping these sites onto the 3?D structure of the human α7 subunit, a structure-function relationship profile was revealed. Our results suggested that the functional divergence related sites clustered in the ligand binding domain, the β2–β3 linker close to the N-terminal α-helix, the intracellular linkers between transmembrane domains, and the “transition zone” may have experienced altered evolutionary rates. The former two regions may be potential binding sites for the α7* subtype-specific allosteric modulators, while the latter region is likely to be subtype-specific allosteric modulations of the heteropentameric descendants such as the α4β2* nAChRs.

Communicated by Ramaswamy H. Sarma