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...

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.

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The auditory hair cell resting potential is critical for proper translation of acoustic signals to the CNS, because it determines their filtering properties, their ability to respond to stimuli of both polarities, and, because the hair cell drives afferent firing rates, the resting potential dictates spontaneous transmitter release. In turtle auditory hair cells, the filtering properties are established by the interactions between BK calcium-activated potassium channels and an L-type calcium channel (electrical resonance). However, both theoretical and in vitro recordings indicate that a third conductance is required to set the resting potential to a point on the I Ca and I BK activation curves in which filtering is optimized like that found in vivo. Present data elucidate a novel mechanism, likely universal among hair cells, in which mechanoelectric transduction (MET) and its calcium-dependent adaptation provide the depolarizing current to establish the hair cell resting potential. First, mechanical block of the MET current hyperpolarized the membrane potential, resulting in broadband asymmetrical resonance. Second, altering steady-state adaptation by altering the [Ca 2ϩ ] bathing the hair bundle changed the MET current at rest, the magnitude of which resulted in membrane potential changes that encompassed the best resonant voltage. The Ca 2ϩ sensitivity of adaptation allowed for the first physiological estimate of endolymphatic Ca 2ϩ near the MET channel (56 Ϯ 11 M), a value similar to bulk endolymph levels. These effects of MET current on resting potential were independently confirmed using a theoretical model of electrical resonance that included the steady-state MET conductance.

Water-soluble models of ligand-gated ion channels would be advantageous for structural studies. We investigated the suitability of three versions of the N-terminal extracellular domain (ECD) of the ␣7 subunit of the nicotinic acetylcholine receptor (AChR) family for this purpose by examining their ligand-binding and assembly properties. Two versions included the first transmembrane domain and were solubilized with detergent after expression in Xenopus oocytes. The third was truncated before the first transmembrane domain and was soluble without detergent. For all three, their equilibrium binding affinities for ␣-bungarotoxin, nicotine, and acetylcholine, combined with their velocity sedimentation profiles, were consistent with the formation of native-like AChRs. These characteristics imply that the ␣7 ECD can form a water-soluble AChR that is a model of the ECD of the full-length ␣7 AChR. Nicotinic acetylcholine receptors (AChRs)1 are integralmembrane, pentameric ion channels in the central and peripheral nervous systems that participate in signal transmission associated with the release of acetylcholine (ACh). A considerable collection of studies of their cell biology, electrophysiology, and structure makes them the best characterized family of a superfamily of homologous neurotransmitter-gated channels that includes glycine, ␥-aminobutyric acid A , and 5-hydroxytryptamine 3 receptors (1-3). Muscle-type AChRs are composed of four different subunits with the subunit composition (␣1) 2 (␤1)␦(␥ or ⑀) and bind the snake venom toxin ␣-bungarotoxin (␣Bgt). Neuronal AChRs that do not bind ␣Bgt are formed from combinations of ␣2, ␣3, ␣4, or ␣6 subunits with ␤2, ␤3, ␤4, and/or ␣5 subunits. Neuronal AChRs that do bind ␣Bgt are formed from ␣7, ␣8, and ␣9 subunits, perhaps in combination with unknown subunits. When heterologously expressed, ␣7, ␣8, and ␣9 form functional homomeric AChRs that appear to contain five identical subunits.AChRs are composed of five homologous membrane-spanning subunits that are ordered around a central, cation-selective channel. The topology of AChRs that is predicted by hydrophobicity plots has received substantial experimental support (4, 5). The approximately 200 residues at the N-terminal half of each AChR subunit are extracellular, are N-glycosylated, contain sites for agonist and antagonist binding, and form the vestibule through which cations reach the transmembrane channel. Relatively little of the remainder of the primary sequence is extracellular. Three of the four transmembrane domains (M1-M3) that form the channel are grouped together following the N-terminal extracellular domain (ECD) and are separated from M4 by a large cytoplasmic loop. For the muscletype AChR, three distinct regions of the primary sequence around amino acid residues 86 -93, 149, and 190 -198 of ␣1 subunits and peptide loops around residues 34, 55-59, 113-119, and 174 -180 of the ␥ or ␦ subunit contribute to the ACh binding sites at the interfaces between ␣ and ␦ and between ␣ and ␥ (or ⑀) subunits, based on phot...

The conformation of methyl A,-(2,2,5,5-tetramethyl-l-oxypyrrolinyl-3-carbonyl)-L-tryptophanate in frozen solutions has been determined by application of electron nuclear double resonance (ENDOR) spectroscopy and computer-based molecular modeling. ENDOR spectra of methyl L-tryptophanate and of the corresponding methyl esters of f3-fluoro-and rj2-fluorotryptophan acylated at the amino group with the spin-label 2,2,5,5-tetramethyl-1 -oxypyrroline-3-carboxylic acid exhibited well-resolved resonance adsorptions from protons and fluorines of the amino acid moiety. The ENDOR shifts were shown to correspond to principal hyperfme coupling (hfc) components, from which the dipolar contributions were estimated to calculate electron-nucleus separations. The ENDOR data indicated that there are two distinct conformations of spin-labeled methyl tryptophanate, the relative populations of which were dependent on solvent polarity. Torsion angle search calculations constrained by the ENDOR data showed that the predominant conformation in methanol was similar to that of a classical % rotamer ( ,--63°) with a near perpendicular ( 2 -(-105°) orientation of the indole ring. The second conformer was characterized by --95°and 2--105°, indicative of an antiperpendicular orientation. In chloroform/toluene only the antiperpendicular conformer was detected. The different solvent-dependent orientations of the indole ring with respect to the nitroxyl group are explained on the basis of dipolar interactions of the aromatic side chain with solvent and with the peptide bond.

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