On the 24 th November 2021 the sequence of a new SARS CoV-2 viral isolate Omicron-B.1.1.529 was announced, containing far more mutations in Spike (S) than previously reported variants. Neutralization titres of Omicron by sera from vaccinees and convalescent subjects infected with early pandemic as well as Alpha, Beta, Gamma, Delta are substantially reduced or fail to neutralize. Titres against Omicron are boosted by third vaccine doses and are high in cases both vaccinated and infected by Delta. Mutations in Omicron knock out or substantially reduce neutralization by most of a large panel of potent monoclonal antibodies and antibodies under commercial development. Omicron S has structural changes from earlier viruses, combining mutations conferring tight binding to ACE2 to unleash evolution driven by immune escape, leading to a large number of mutations in the ACE2 binding site which rebalance receptor affinity to that of early pandemic viruses.

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 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.

A mutation (S247F) in the channel-lining domain (M2) of the ␣4 nicotinic acetylcholine receptor (AChR) subunit has previously been linked genetically to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE).To better understand the functional significance of this mutation, we characterized the properties of mutant and wild-type human ␣4␤2 AChRs expressed in Xenopus oocytes. Both had similar expression levels and EC 50 values for ACh and nicotine. Substantial use-dependent functional upregulation was found for mutant ␣4␤2 AChRs, but not for wild type. Mutant AChR responses showed faster desensitization, slower recovery from desensitization, less inward rectification, and virtually no Ca 2ϩ permeability as compared with wild-type ␣4␤2 AChRs. Addition of the ␣5 subunit restored Ca 2ϩ permeability to the mutant ␣4␤2␣5 AChRs. At Ϫ80 mV, wild-type ␣4␤2 AChR single channel currents exhibited two conductances, each with two mean open times (␥ 1 ϭ 17 pS, 1 ϭ 3.7 msec, and 2 ϭ 23.4 msec; ␥ 2 ϭ 28 pS, 1 ϭ 1.9 msec, and 2 ϭ 8.1 msec). In contrast, mutant AChRs exhibited only one conductance of 11 pS, with 1 ϭ 1.9 msec and 2 ϭ 4.1 msec.The net effect of the mutation is to reduce AChR function. This could result in the hyperexcitability characteristic of epilepsy if the mutant AChRs were part of an inhibitory circuit, e.g., presynaptically regulating the release of GABA. In the minority of AChRs containing the ␣5 subunit, the overall functionality of these AChRs could be maintained despite the mutation in the ␣4 subunit.