Two functional types of nicotinic acetylcholine receptors (nAChRs) are expressed when human embryonic kidney cells are permanently transfected with equal amounts of human ␣4 and ␤2 subunit cDNAs. Most (82%) of these nAChRs exhibit an EC 50 of 74 Ϯ 6 M for ACh, a much lower sensitivity than the remaining fraction (EC 50 of 0.7 Ϯ 0.4 M) or than expected from expression of equal amounts of ␣4 and ␤2 mRNAs in Xenopus laevis oocytes. We have found three conditions that can increase the number of nAChRs with high sensitivity to activation. These are: 1) transient transfection with additional ␤2 subunits, 2) overnight incubation in nicotine, or 3) overnight culture at 29°C. Using metabolic labeling with [ 35 S]methionine to measure subunit stoichiometry, we found that the majority of nAChRs had a stoichiometry of (␣4) 3 (␤2) 2 . Overnight treatment with nicotine increased the number of nAChRs and increased the proportion of the (␣4) 2 (␤2) 3 stoichiometry. Alternate ␣4␤2 nAChR stoichiometries with distinct functional properties raise the possibility for an interesting mode of synaptic regulation for nicotinic signaling in the mammalian brain.The ␣4␤2 nAChR is the predominant nAChR subtype in the mammalian brain that has high affinity for nicotine. nAChRs composed of ␣4 and ␤2 subunits modulate neurotransmitter release (Dani, 2001) and play a direct role in addiction to nicotine (Picciotto et al., 1998;Marubio et al., 1999). Mutations in ␣4␤2 nAChRs have been linked to autosomal-dominant nocturnal frontal lobe epilepsy (Weiland et al., 2000). They also are thought to be involved in Alzheimer's and Parkinson's diseases (Rusted et al., 2000).Two different approaches showed independently that chick ␣4␤2 nAChRs have a stoichiometry of (␣4) 2 (␤2) 3 when expressed in Xenopus laevis oocytes from cRNAs or cDNAs injected at a 1:1 (␣/␤) ratio (Anand et al., 1991;Cooper et al., 1991). A more recent study showed that when the rat ␣4/␤2 subunit ratio is varied, nAChRs of two functional classes are formed in oocytes (Zwart and Vijverberg, 1998). When the ␣4/␤2 ratio was 1:9, nAChRs were formed that were more sensitive to activation and desensitized more slowly. However, when the ratios were 1:1 or 9:1, nAChRs appeared that were less sensitive to activation and desensitized more rapidly. These findings raised the possibility that ␣4␤2 nAChRs can also exist in a stoichiometry that differs from (␣4) 2 (␤2) 3 .Here, we report that the majority (82%) of ␣4␤2 nAChRs expressed in a stable HEK cell line exhibit sensitivity to activation by ACh that is much lower (EC 50 ϭ 74 Ϯ 6 M) than when ␣4␤2 nAChRs are expressed in Xenopus laevis , 1997)]. This confirms the observations by Buisson and Bertrand (2001) in another independently derived line. They also reported that nicotine and other nicotinic agents increased the proportion of high-sensitivity nAChRs and speculated that the increase was caused by slow conversion of existing low-sensitivity nAChRs to high-sensitivity nAChRs. However, it is well known that nicotine, other nicotinic ...

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.

Understanding effects of chronic nicotine requires identifying the neurons and synapses whose responses to nicotine itself, and to endogenous acetylcholine, are altered by continued exposure to the drug. To address this problem, we developed mice whose ␣4 nicotinic receptor subunits are replaced by normally functioning fluorescently tagged subunits, providing quantitative studies of receptor regulation at micrometer resolution. Chronic nicotine increased ␣4 fluorescence in several regions; among these, midbrain and hippocampus were assessed functionally. Although the midbrain dopaminergic system dominates reward pathways, chronic nicotine does not change ␣4* receptor levels in dopaminergic neurons of ventral tegmental area (VTA) or substantia nigra pars compacta. Instead, upregulated, functional ␣4* receptors localize to the GABAergic neurons of the VTA and substantia nigra pars reticulata. In consequence, GABAergic neurons from chronically nicotine-treated mice have a higher basal firing rate and respond more strongly to nicotine; because of the resulting increased inhibition, dopaminergic neurons have lower basal firing and decreased response to nicotine. In hippocampus, chronic exposure to nicotine also increases ␣4* fluorescence on glutamatergic axons of the medial perforant path. In hippocampal slices from chronically treated animals, acute exposure to nicotine during tetanic stimuli enhances induction of long-term potentiation in the medial perforant path, showing that the upregulated ␣4* receptors in this pathway are also functional. The pattern of cell-specific upregulation of functional ␣4* receptors therefore provides a possible explanation for two effects of chronic nicotine: sensitization of synaptic transmission in forebrain and tolerance of dopaminergic neuron firing in midbrain.

Autoantibodies are frequently observed in healthy individuals. In a minority of these individuals, they lead to manifestation of autoimmune diseases, such as rheumatoid arthritis or Graves' disease. Overall, more than 2.5% of the population is affected by autoantibody-driven autoimmune disease. Pathways leading to autoantibody-induced pathology greatly differ among different diseases, and autoantibodies directed against the same antigen, depending on the targeted epitope, can have diverse effects. To foster knowledge in autoantibody-induced pathology and to encourage development of urgently needed novel therapeutic strategies, we here categorized autoantibodies according to their effects. According to our algorithm, autoantibodies can be classified into the following categories: (1) mimic receptor stimulation, (2) blocking of neural transmission, (3) induction of altered signaling, triggering uncontrolled (4) microthrombosis, (5) cell lysis, (6) neutrophil activation, and (7) induction of inflammation. These mechanisms in relation to disease, as well as principles of autoantibody generation and detection, are reviewed herein.

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