Human neuronal nicotinic acetylcholine receptor (AChR) alpha4 subunits and an alpha4 mutant (S247Falpha4) found in autosomal-dominant nocturnal frontal lobe epilepsy (ADNFLE) were expressed along with beta2 in permanently transfected tsA201 human embryonic kidney cell lines. Their sensitivity to activation, desensitization, and up-regulation by cholinergic ligands was investigated. Up-regulation after 3 to 24 h resulted primarily from an increase in assembly of AChRs from large pools of unassembled subunits, but up-regulation also resulted from a 5-fold increase in the lifetime of AChRs in the surface membrane. Up-regulation does not require current flow through surface membrane AChRs, because up-regulation occurs in the presence of the channel blocker mecamylamine and with the alpha4 mutant, which prevents nearly all AChR function. Both membrane-permeable ligands like nicotine and much less permeable quaternary amine cholinergic ligands can act as pharmacological chaperones within the endoplasmatic reticulum to promote the assembly of AChRs. Agonists are more potent pharmacological chaperones than antagonists, presumably because activated or desensitized conformations assemble more efficiently. Assembly intermediates are disrupted by solubilization in Triton X-100, but chemical cross-linking stabilizes a putative assembly intermediate approximately the size of an alpha4beta2alpha4beta2 tetramer.

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Human nicotinic acetylcholine receptor (AChR) subtypes ␣3␤2, ␣3␤2␣5, ␣3␤4, and ␣3␤4␣5 were stably expressed in cells derived from the human embryonic kidney cell line 293. ␣3␤4 AChRs were found in prominent 2-m patches on the cell surface, whereas most ␣3␤2 AChRs were more diffusely distributed. The functional properties of the ␣3 AChRs in tsA201 cells were characterized by whole cell patch clamp using both acetylcholine and nicotine as agonists. Nicotine was a partial agonist on ␣3␤4 AChRs and nearly a full agonist on ␣3␤2␣5 AChRs. Chronic exposure of cells expressing ␣3␤2 AChRs or ␣3␤2␣5 AChRs to nicotine or carbamylcholine increased their amount up to 24-fold but had no effect on the amount of ␣3␤4 or ␣3␤4␣5 AChRs, i.e. the up-regulation of ␣3 AChRs depended on the presence of ␤2 but not ␤4 subunits in the AChRs. This was also found to be true of ␣3 AChRs in the human neuroblastoma SH-SY5Y. In the absence of nicotine, ␣3␤2 AChRs were expressed at much lower levels than ␣3␤4 AChRs, but in the presence of nicotine, the amount of ␣3␤2 AChRs exceeded that of ␣3␤4 AChRs. Up-regulation was seen for both total AChRs and surface AChRs. Up-regulated ␣3␤2 AChRs were functional. The nicotinic antagonists curare and dihydro-␤-erythroidine also up-regulated ␣3␤2 AChRs, but only by 3-5-fold. The channel blocker mecamylamine did not cause up-regulation of ␣3␤2 AChRs and inhibited up-regulation by nicotine. Our data suggest that up-regulation of ␣3␤2 AChRs in these lines by nicotine results from both increased subunit assembly and decreased AChR turnover.

We prepared concatamers of alpha4 and beta2 subunits for human nicotinic acetylcholine receptors (AChRs), in which the C terminus of alpha4 was linked to the N terminus of beta2, 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 alpha4 prevented AChR potentiation by 17-beta-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 (alpha4)3(beta2)2 stoichiometry than with the (alpha4)2(beta2)3 stoichiometry. The (alpha4)3(beta2)2 stoichiometry exhibited much lower ACh sensitivity. When concatamers were expressed alone, dipentameric AChRs were formed in which the (alpha4)2(beta2)3 pentamer was linked to the (alpha4)3(beta2)2 pentamer. Dipentamers were selectively expressed on the cell surface, whereas most monopentamers with dangling subunits were retained intracellularly. Coexpression of concatamers with monomeric beta2, beta4, or alpha4 subunits resulted in monopentamers, the stoichiometry of which was determined by the free subunit added. Linkage between the C terminus of beta2 and the N terminus of alpha4 favored formation of ACh-binding sites within the concatamer, whereas linkage between the C terminus of alpha4 and the N terminus of beta2 favored formation of ACh-binding sites between concatamers. These protein-engineering studies provide insight into the structure and function of alpha4beta2 AChRs, emphasizing the functional differences between alpha4beta2 AChRs of different stoichiometries.

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Selected nicotinic agonists were used to activate and desensitize high-sensitivity (HS) (a4) 2 (b2) 3 ) or low-sensitivity (LS) (a4) 3 (b2) 2 ) isoforms of human a4b2-nicotinic acetylcholine receptors (nAChRs). Function was assessed using 86 Rb1 efflux in a stably transfected SH-EP1-ha4b2 human epithelial cell line, and twoelectrode voltage-clamp electrophysiology in Xenopus laevis oocytes expressing concatenated pentameric HS or LS a4b2-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 a4b2-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 a4(1)/(2)a4 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 b2-subunit-specific [ 125 I]mAb295 labeling. However, maximum function is approximately five times greater on a "per-receptor" basis for unmodified LSP versus HSP a4b2-nAChRs. Thus, recruitment of the a4(1)/(2)a4 site at higher agonist concentrations appears to augment otherwisesimilar function mediated by the pair of a4(1)/(2)b2 sites shared by both isoforms. These studies elucidate the receptor-level differences underlying the differential pharmacology of the two a4b2-nAChR isoforms, and demonstrate that HS versus LS a4b2-nAChR activity can be selectively manipulated using pharmacological approaches. Since a4b2 nAChRs are the predominant neuronal subtype, these discoveries likely have significant functional implications, and may provide important insights for drug discovery and development.

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A recent report described the isolation of cDNA clones encoding alpha 7 and alpha 8 subunits of alpha-bungarotoxin-sensitive nicotinic ACh receptors (alpha BgtAChRs) from chick brain and demonstrated that they were related to, but distinct from, the alpha subunits of nicotinic ACh receptors (nAChRs) from muscles and neurons. Monoclonal antibodies against the two alpha BgtAChR subunits were used to demonstrate that at least two subtypes are present in embryonic day 18 chicken brain. The predominant brain subtype contains alpha 7 subunits, while a minor subtype contains both alpha 7 and alpha 8 subunits. Both subtypes may also contain other subunits. Here we report the results of immune precipitation studies and immunohistochemical studies of alpha BgtAChRs in the chick retina. In addition to the two subtypes found in brain, a new alpha BgtAChR subtype that contains alpha 8 subunits, but not alpha 7 subunits, was identified and was found to be the major subtype in chick retina. This subtype has a lower affinity for alpha-bungarotoxin (alpha Bgt) than does the subtype containing only alpha 7 subunits. Small amounts of this alpha 8 subtype were also detected in brain by labeling with higher concentrations of 125I-alpha Bgt than had been used previously. The subtype containing only alpha 7 subunits comprised 14% of the alpha BgtAChRs in hatchling chick retina. The subtype containing alpha 8 subunits (but no alpha 7 subunits) accounted for 69%, and the alpha 7 alpha 8 subtype accounted for 17%. Amacrine, bipolar, and ganglion cells displayed alpha 8 subunit immunoreactivity, and a complex pattern of labeling was evident in both the inner and outer plexiform layers. In contrast, only amacrine and ganglion cells exhibited alpha 7 subunit immunoreactivity, and the pattern of alpha 7 subunit labeling in the inner plexiform layer differed from that of alpha 8 subunit labeling. These disparities suggest that the alpha BgtAChR subunits are differentially expressed by different populations of retinal neurons. In addition, the distribution of alpha BgtAChR subunit immunoreactivity was found to differ from that of alpha-Bgt-insensitive nAChR subunits.