Nicotinic acetylcholine receptors formed from combinations of ␣3, ␤2, ␤4, and ␣5 subunits are found in chicken ciliary ganglion neurons and some human neuroblastoma cell lines. We studied the co-expression of various combinations of cloned human ␣3, ␤2, ␤4, and ␣5 subunits in Xenopus oocytes. Expression on the surface membrane was found only for combinations of ␣3␤2, ␣3␤4, ␣3␤2␣5, and ␣3␤4␣5 subunits but not for other combinations of one, two, or three of these subunits. ␣5 subunits assembled inside the oocyte with ␤2 but not with ␣3 subunits or other ␣5 subunits. ␣5 subunits coassembled very efficiently with ␣3␤2 or ␣3␤4 combinations. The presence of ␣5 subunits had very little effect on the binding affinities for epibatidine of receptors containing also ␣3 and ␤2 or ␣3 and ␤4 subunits. The presence of ␣5 subunits increased the rate of desensitization of both receptors containing also ␣3 and ␤2 or ␣3 and ␤4 subunits. In the case of receptors containing ␣3 and ␤4 subunits, the addition of ␣5 subunits had little effect on the responses to acetylcholine or nicotine. However, in the case of receptors containing ␣3 and ␤2 subunits, the addition of ␣5 subunits reduced the EC 50 for acetylcholine from 28 to 0.5 M and the EC 50 for nicotine from 6.8 to 1.9 M, while increasing the efficacy of nicotine from 50% on ␣3␤2 receptors to 100% on ␣3␤2␣5 receptors. Both ␣3␤2 and ␣3␤2␣5 receptors expressed in oocytes sedimented at the same 11 S value as native ␣3-containing receptors from the human neuroblastoma cell line SH-SY5Y. In the receptors from the neuroblastoma ␣3, ␤2, and ␣5 subunits were co-assembled, and 56% of the receptor subtypes containing ␣3 subunits also contained ␤2 subunits. The ␤2 subunitcontaining receptors from SH-SY5Y cells exhibited the high affinity for epibatidine characteristic of receptors formed from ␣3 and ␤2 or ␣3, ␤2, and ␣5 subunits rather than the low affinity exhibited by receptors formed from ␣3 and ␤4 or ␣3, ␤4, and ␣5 subunits. Nicotine, like the structurally similar toxin epibatidine, also distinguishes by binding affinity two subtypes of receptors containing ␣3 subunits in SH-SY5Y cells. The affinities of ␣3␤2 receptors expressed in oocytes were similar to the affinities of native ␣3 containing receptors from SH-SY5Y cells for acetylcholine, cytisine, and 1,1-dimethyl-4-phenylpiperazinium. Nicotinic acetylcholine receptors (AChRs)1 are members of a gene superfamily of homologous ligand-gated ion channels which include receptors for glycine, ␥-aminobutyric acid, and serotonin (1). There are three branches of the AChR gene family (2-5). The best characterized are muscle and electric organ AChRs which consist of a pentameric array of homologous subunits oriented around a central ion channel like barrel staves. The order of these subunits around the channel is ␣1␥␣1␦␤1 in the fetal form and ␣1⑀␣1␦␤1 in the adult form (6). The two ligand binding sites in each AChR are thought to be formed at the interfaces between ␣1 and ␥, ␦, or ⑀ subunits (6). One group of neuronal AChRs which is capable of f...

Chronic exposure to nicotine has been reported to increase the number of nicotinic acetylcholine receptors (AChRs) in brain. The mechanism of up-regulation for the alpha4beta2 AChR subtype, which accounts for the majority of high affinity nicotine binding in mammalian brain, has previously been shown to involve a decrease in the rate of alpha4beta2 AChR turnover. Here, we report an investigation of the extent and mechanism of nicotine-induced up-regulation of alpha3 AChRs and alpha7 AChR subtypes expressed in the human neuroblastoma cell line SH-SY5Y. Up-regulation of human alpha3 AChRs and alpha7 AChRs, unlike alpha4beta2 AChRs, requires much higher nicotine concentrations than are encountered in smokers; the extent of increase of surface AChRs is much less; and the mechanisms of up-regulation are different than with alpha4beta2 AChRs. The mechanisms of up-regulation may be different for alpha3 AChRs or alpha7 AChRs. Chronic treatment with nicotine or carbamylcholine, but not d-tubocurarine, mecamylamine, or dihydro-beta-erythroidine, induced a 500-600% increase in the number of alpha3 AChRs but only a 30% increase in alpha7 AChRs. Chronic nicotine treatment did not increase affinity for nicotine or increase the amount of RNA for alpha3 or alpha7 subunits. The effect of nicotine on up-regulation of alpha7 AChRs was partially blocked by either d-tubocurarine or mecamylamine. The effect of nicotine treatment on the number of alpha3 AChRs was only slightly blocked by the antagonists d-tubocurarine, mecamylamine, or dihydro-beta-erythroidine at concentrations that efficiently block alpha3 AChR function. Most of the nicotine-induced increase in alpha3 AChRs was found to be intracellular. The alpha3 AChRs, which accumulate intracellularly, were shown to have been previously exposed on the cell surface by their susceptibility to antigenic modulation. The data suggest that chronic exposure to nicotine may induce a conformation of cell surface alpha3 AChRs that at least in this cell line are consequently internalized but not immediately destroyed.

Little is known about the genetic basis of convergent traits that originate repeatedly over broad taxonomic scales. The myogenic electric organ has evolved six times in fishes to produce electric fields used in communication, navigation, predation, or defense. We have examined the genomic basis of the convergent anatomical and physiological origins of these organs by assembling the genome of the electric eel (Electrophorus electricus) and sequencing electric organ and skeletal muscle transcriptomes from three lineages that have independently evolved electric organs. Our results indicate that, despite millions of years of evolution and large differences in the morphology of electric organ cells, independent lineages have leveraged similar transcription factors and developmental and cellular pathways in the evolution of electric organs.

By using the large cytoplasmic domain of the nicotinic acetylcholine receptor (AChR) ␣4 subunit as a bait in the yeast two-hybrid system, we isolated the first cytosolic protein, 14-3-3, known to interact directly with neuronal AChRs. 14-3-3 is a member of a family of proteins that function as regulatory or chaperone/ scaffolding/adaptor proteins. 14-3-3 interacted with the recombinant ␣4 subunit alone in tsA 201 cells following activation of cAMP-dependent protein kinase by forskolin. The interaction of 14-3-3 with recombinant ␣4 subunits was abolished when serine 441 of the ␣4 subunit was mutated to alanine (␣4 S441A ). The surface levels of recombinant wild-type ␣4␤2 AChRs were ϳ2-fold higher than those of mutant ␣4 S441A ␤2 AChRs. The interaction significantly increased the steady state levels of the ␣4 subunit and ␣4␤2 AChRs but not that of the mutant ␣4 S441A subunit or mutant ␣4 S441A ␤2 AChRs. The EC 50 values for activation by acetylcholine were not significantly different for ␣4␤2 AChRs and ␣4 S441A ␤2 AChRs coexpressed with 14-3-3 in oocytes following treatment with forskolin. 14-3-3 coimmunopurified with native ␣4 AChRs from brain. These results support a role for 14-3-3 in dynamically regulating the expression levels of ␣4␤2 AChRs through its interaction with the ␣4 subunit. Neuronal nicotinic acetylcholine receptors (AChR)1 are a family of ligand-gated, cation-selective, homo-or heteropentameric ion channels expressed in the peripheral and central nervous system (1, 2). A multitude of neuronal AChR subtypes assembled from different combinations of ␣2-␣9 and ␤2-␤4 subunits have been identified (3,4 (7), and show attenuated self-administration of nicotine (8) suggesting that ␣4␤2 AChRs have a role in mediating addiction to nicotine. The normal and pathophysiological functions mediated by ␣4␤2 AChRs are of significant importance to human health. Some inherited forms of epilepsy, such as the autosomal dominant nocturnal frontal lobe epilepsies, are caused by ␣4␤2 AChRs harboring at least two separate mutations within their ␣4 subunit (9 -12). Most recently, ␣4␤2 AChRs, among other ␤2 subunit-containing AChRs, have been implicated in neuronal survival during aging, as surmised from the neurodegeneration observed in ␤2-subunit knock-out mice (13).The ␣4 subunit, like the other AChR subunits, consists of an extracellular N-terminal domain, followed by three transmembrane domains (M1-M3), a large cytoplasmic domain, a fourth transmembrane domain (M4), and a short extracellular C terminus. The large cytoplasmic domain is highly divergent among the various subunits, and this sequence divergence presumably provides the diversity necessary for different AChR subtypes to interact directly with cytosolic proteins of different function. To identify such proteins associated with ␣4␤2 AChRs, we used the large cytoplasmic domain of the ␣4 subunit as a bait to screen a mouse brain cDNA yeast two-hybrid library. Here we describe the isolation of a known protein termed 14-3-3. The 14-3-3 proteins family consists of sev...

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