Alexander Kuryatov

Prenatal nicotine increases pulmonary α7 nicotinic receptor expression and alters fetal lung development in monkeys

It is well established that maternal smoking during pregnancy is a leading preventable cause of low birth weight and prematurity. Less appreciated is that maternal smoking during pregnancy is also associated with alterations in pulmonary function at birth and greater incidence of respiratory illnesses after birth. To determine if this is the direct result of nicotine interacting with nicotinic cholinergic receptors (nAChRs) during lung development, rhesus monkeys were treated with 1 mg/kg/day of nicotine from days 26 to 134 of pregnancy. Nicotine administration caused lung hypoplasia and reduced surface complexity of developing alveoli. Immunohistochemistry and in situ alpha-bungarotoxin (alphaBGT) binding showed that alpha7 nAChRs are present in the developing lung in airway epithelial cells, cells surrounding large airways and blood vessels, alveolar type II cells, free alveolar macrophages, and pulmonary neuroendocrine cells (PNEC). As detected both by immunohistochemistry and by alphaBGT binding, nicotine administration markedly increased alpha7 receptor subunit expression and binding in the fetal lung. Correlating with areas of increased alpha7 expression, collagen expression surrounding large airways and vessels was significantly increased. Nicotine also significantly increased numbers of type II cells and neuroendocrine cells in neuroepithelial bodies. These findings demonstrate that nicotine can alter fetal monkey lung development by crossing the placenta to interact directly with nicotinic receptors on non-neuronal cells in the developing lung, and that similar effects likely occur in human infants whose mothers smoke during pregnancy.

Human α6 AChR subtypes : subunit composition, assembly, and pharmacological responses

Many nicotinic acetylcholine receptor (AChR) subunits are known to be co-expressed with the alpha6 subunit in neurons. Because alpha6beta4 AChRs assemble inefficiently and alpha6beta2 AChRs not at all, more complex mixtures of human subunit cDNAs were tested for their abilities to form functional AChRs when expressed in Xenopus oocytes. alpha6beta4beta3 AChRs produced the largest and most consistent responses. alpha6alpha3beta2 AChRs exhibited reduced potency for ACh and increased potency and efficacy for nicotine compared to alpha3beta2 AChRs, but similar resistance to functional inactivation after prolonged exposure to nicotine. alpha6alpha4beta2 AChRs differed little in potency or efficacy for ACh or nicotine compared to alpha4beta2 AChRs, and had similarly high sensitivity to inactivation by prolonged exposure to nicotine. Co-expression of alpha6 and beta2 cRNAs resulted in large numbers of (3)H-epibatidine binding sites in the form of large aggregates but not in functional pentameric AChRs. Co-expression of alpha6, beta2, and alpha5 resulted in assembly of some functional pentameric AChRs. Chimeras with the large extracellular domain of alpha6 and the rest from either alpha3 or alpha4 efficiently formed functional AChRs. Thus, the extracellular domain of alpha6 efficiently assembles with beta2 to form ACh binding sites, but more C-terminal domains cause difficulties in forming pentameric AChRs. Chimeric alpha6/alpha3 and alpha6/alpha4 AChRs containing either beta2 or beta4 subunits were blocked by alpha-conotoxin MII which had previously been reported to be specific for alpha3beta2 AChRs.

Chronic Nicotine Treatment Up-regulates Human α3β2 but Not α3β4 Acetylcholine Receptors Stably Transfected in Human Embryonic Kidney Cells

Human nicotinic acetylcholine receptor (AChR) subtypes α3β2, α3β2α5, α3β4, and α3β4α5 were stably expressed in cells derived from the human embryonic kidney cell line 293. α3β4 AChRs were found in prominent 2-μm patches on the cell surface, whereas most α3β2 AChRs were more diffusely distributed. The functional properties of the α3 AChRs in tsA201 cells were characterized by whole cell patch clamp using both acetylcholine and nicotine as agonists. Nicotine was a partial agonist on α3β4 AChRs and nearly a full agonist on α3β2α5 AChRs. Chronic exposure of cells expressing α3β2 AChRs or α3β2α5 AChRs to nicotine or carbamylcholine increased their amount up to 24-fold but had no effect on the amount of α3β4 or α3β4α5 AChRs, i.e. the up-regulation of α3 AChRs depended on the presence of β2 but not β4 subunits in the AChRs. This was also found to be true of α3 AChRs in the human neuroblastoma SH-SY5Y. In the absence of nicotine, α3β2 AChRs were expressed at much lower levels than α3β4 AChRs, but in the presence of nicotine, the amount of α3β2 AChRs exceeded that of α3β4 AChRs. Up-regulation was seen for both total AChRs and surface AChRs. Up-regulated α3β2 AChRs were functional. The nicotinic antagonists curare and dihydro-β-erythroidine also up-regulated α3β2 AChRs, but only by 3–5-fold. The channel blocker mecamylamine did not cause up-regulation of α3β2 AChRs and inhibited up-regulation by nicotine. Our data suggest that up-regulation of α3β2 AChRs in these lines by nicotine results from both increased subunit assembly and decreased AChR turnover.

Roles of Accessory Subunits in α4β2* Nicotinic Receptors

Accessory subunits in heteromeric nicotinic receptors (AChRs) do not take part in forming ACh binding sites. α5 and β3 subunits can function only as accessory subunits. We show that both α5 and β3 efficiently assemble in human α4β2* AChRs expressed in permanently transfected human embryonic kidney (HEK) cell lines. Only (α4β2)2α5, not (α4β2)2β3 AChRs, have been detected in brain. The α4β2α5 line expressed 40% more AChRs than the parent α4β2 line and was equally sensitive to up-regulation by nicotine. The α4β2β3 line expressed 25-fold more AChRs than the parental line and could not be further up-regulated by nicotine. Relative sensitivity to activation by ACh depends on the accessory subunit, β2 conferring the greatest sensitivity, α5 less, and β3 and α4 much less. Accessory subunits form binding sites for positive allosteric modulators, as illustrated by the observation that α5 conferred high sensitivity to galanthamine. In the presence of α5 or β3, stable, partially degraded, dead end intermediates accumulated within the cells. These may have the form α5α4β2α5. The efficiency with which α5 and β3 assemble with α4 and β2 and the necessity of avoiding formation of potentially toxic intermediates may explain why α5 and β3 seem to be transcribed at low levels in brain. Autosomal dominant nocturnal frontal lobe epilepsy can be caused by the α4 mutation S247F. This mutant did not produce functional AChRs unless cells were cotransfected with α5, β3, or α6 to replace α4 as accessory subunit.

Ca2+ Permeability of the (α4)3(β2)2 Stoichiometry Greatly Exceeds That of (α4)2(β2)3 Human Acetylcholine Receptors

Human α4β2 nicotinic acetylcholine receptors (AChRs) expressed in Xenopus laevis oocytes or transfected cell lines are present as a mixture of two stoichiometries, (α4)2(β2)3 and (α4)3(β2)2, which differ depending on whether a β2 or α4 subunit occupies the accessory subunit position corresponding to β1 subunits of muscle AChRs. Pure populations of each stoichiometry can be expressed in oocytes by combining a linked pair of α4 and β2 with free β2 to produce the (α4)2(β2)3 stoichiometry or with free α4 to produce the (α4)3(β2)2 stoichiometry. We show that the (α4)3(β2)2 stoichiometry and the (α4)2(β2)2β3 and (α4)2(β2)2α5 subtypes in which β3 or α5occupy the accessory positions have much higher permeability to Ca2+ than does (α4)2(β2)3 and suggest that this could be physiologically significant in triggering signaling cascades if this stoichiometry or these subtypes were found in vivo. We show that Ca2+ permeability is determined by charged amino acids at the extracellular end of the M2 transmembrane domain, which could form a ring of amino acids at the outer end of the cation channel. α4, α5, and β3 subunits all have a homologous glutamate in M2 that contributes to high Ca2+ permeability, whereas β2 has a lysine at this position. Subunit combinations or single amino acids changes at this ring that have all negative charges or a mixture of positive and negative charged amino acids are permeable to Ca2+. All positive charges in the ring prevent Ca2+ permeability. Increasing the proportion of negative charges is associated with increasing permeability to Ca2+.

Human α4β2 Acetylcholine Receptors Formed from Linked Subunits

We prepared concatamers of α4 and β2 subunits for human nicotinic acetylcholine receptors (AChRs), in which the C terminus of α4 was linked to the N terminus of β2, or vice versa, via a tripeptide sequence repeated 6 or 12 times, and expressed them in Xenopus oocytes. Linkage did not substantially alter channel amplitude or channel open-duration. Linkage at the C terminus of α4 prevented AChR potentiation by 17-β-estradiol by disruption of its binding site. Assembly of AChRs from concatamers was less efficient, but function was much more efficient than that of unlinked subunits. With both linked and free subunits, greater ACh-induced currents per surface AChR were observed with the (α4)3(β2)2 stoichiometry than with the (α4)2(β2)3 stoichiometry. The (α4)3(β2)2 stoichiometry exhibited much lower ACh sensitivity. When concatamers were expressed alone, dipentameric AChRs were formed in which the (α4)2(β2)3 pentamer was linked to the (α4)3(β2)2 pentamer. Dipentamers were selectively expressed on the cell surface, whereas most monopentamers with dangling subunits were retained intracellularly. Coexpression of concatamers with monomeric β2, β4, or α4 subunits resulted in monopentamers, the stoichiometry of which was determined by the free subunit added. Linkage between the C terminus of β2 and the N terminus of α4 favored formation of ACh-binding sites within the concatamer, whereas linkage between the C terminus of α4 and the N terminus of β2 favored formation of ACh-binding sites between concatamers. These protein-engineering studies provide insight into the structure and function of α4β2 AChRs, emphasizing the functional differences between α4β2 AChRs of different stoichiometries.

Acetylcholine Receptor (AChR) α5 Subunit Variant Associated with Risk for Nicotine Dependence and Lung Cancer Reduces (α4β2)2α5 AChR Function

Genomic studies have identified a D398N variation in the α5 subunit of nicotinic acetylcholine receptors (AChRs) that increases risk of nicotine dependence and lung cancer. (α4β2)2α5 AChRs are a significant brain presynaptic subtype in brain. Their high sensitivity to activation by nicotine and high Ca2+ permeability give them substantial functional impact. α3β4* and α3β2* AChRs are predominant postsynaptic AChRs in the autonomic nervous system, but rare in brain. The amino acid 398 of α5 is located in the large cytoplasmic domain near the amphipathic α helix preceding the M4 transmembrane domain. These helices have been shown to influence AChR conductance by forming portals to the central channel. We report that α5 Asn 398 lowers Ca2+ permeability and increases short-term desensitization in (α4β2)2α5 but not in (α3β4)2α5 or (α3β2)2α5 AChRs. This suggests that a positive allosteric modulator would augment nicotine replacement therapy for those with this risk variant. α5 D398N variation does not alter sensitivity to activation. The high sensitivity to activation and desensitization of (α4β2)2α5 AChRs by nicotine results in a narrow concentration range in which activation and desensitization curves overlap. This region centers on 0.2 μM nicotine, a concentration typically sustained in smokers. This concentration would desensitize 60% of these AChRs and permit smoldering activation of the remainder. The low sensitivity to activation and desensitization of (α3β4)2α5 AChRs by nicotine results in a broad region of overlap centered near 10 μM. Thus, at the nicotine concentrations in smokers, negligible activation or desensitization of this subtype would occur.

β3 Subunits Promote Expression and Nicotine-Induced Up-Regulation of Human Nicotinic α6* Nicotinic Acetylcholine Receptors Expressed in Transfected Cell Lines

Nicotinic acetylcholine receptors (AChRs) containing α6 subunits are typically found at aminergic nerve endings where they play important roles in nicotine addiction and Parkinsons disease. α6* AChRs usually contain β3 subunits. β3 subunits are presumed to assemble only in the accessory subunit position within AChRs where they do not participate in forming acetylcholine binding sites. Assembly of subunits in the accessory position may be a critical final step in assembly of mature AChRs. Human α6 AChRs subtypes were permanently transfected into human tsA201 human embryonic kidney (HEK) cell lines. α6β2β3 and α6β4β3 cell lines were found to express much larger amounts of AChRs and were more sensitive to nicotine-induced increase in the amount of AChRs than were α6β2 or α6β4 cell lines. The increased sensitivity to nicotine-induced up-regulation was due not to a β3-induced increase in affinity for nicotine but probably to a direct effect on assembly of AChR subunits. HEK cells express only a small amount of mature α6β2 AChRs, but many of these subunits are on the cell surface. This contrasts with Xenopus laevis oocytes, which express a large amount of incorrectly assembled α6β2 subunits that bind cholinergic ligands but form large amorphous intracellular aggregates. Monoclonal antibodies (mAbs) were made to the α6 and β3 subunits to aid in the characterization of these AChRs. The α6 mAbs bind to epitopes C-terminal of the extracellular domain. These data demonstrate that both cell type and the accessory subunit β3 can play important roles in α6* AChR expression, stability, and up-regulation by nicotine.

Expression of Functional Human α6β2β3* Acetylcholine Receptors in Xenopus laevis Oocytes Achieved through Subunit Chimeras and Concatamers

α6β2β3* acetylcholine receptors (AChRs) on dopaminergic neurons are important targets for drugs to treat nicotine addiction and Parkinsons disease. However, it has not been possible to efficiently express functional α6β2β3* 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 α6β2β3* AChRs with defined subunit compositions and subunit orders. Assembly of accessory subunits is limiting in formation of mature AChRs. A single linker between the β3 accessory subunit and an α4 or α6 subunit is sufficient to permit assembly of complex β3-(α4β2)(α6β2) or β3-(α6β2)(α4β2) AChRs. Concatameric pentamers such as β3-α6-β2-α4-β2 have been functionally characterized. α6β2β3* AChRs are sensitive to activation by drugs used for smoking cessation therapy (nicotine, varenicline, and cytisine) and by sazetidine. All these are partial agonists. (α6β2)(α4β2)β3 AChRs are most sensitive to agonists. (α6β2)2β3 AChRs have the greatest Ca2+ permeability. (α4β2)(α6β2)β3 AChRs are most efficiently transported to the cell surface, whereas (α6β2)2β3 AChRs are the least efficiently transported. Dopaminergic neurons may have special chaperones for assembling accessory subunits with α6 subunits and for transporting (α6β2)2β3 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 α6β2β3* AChRs. These concatamers should enable selection of drugs specific for α6β2β3* AChRs.

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.