Phylogenetic conservation of disulfide-linked, dimeric acetylcholine receptor pentamers in southern ocean electric rays

Abstract

Intact acetylcholine receptors have been purified on a novel affinity resin from three electric fish endemic to Australian waters. Their binding properties and morphology are compared with those of their northern hemisphere homolog, Torpedo marmorata. All four exhibit apparent dissociation constants, Kd, in the nanomolar range for the snake neurotoxin α-bungarotoxin and have a distinctive rosette-like appearance when viewed in negative stain under the electron microscope. Furthermore, these rosettes are paired, indicating that acetylcholine receptors from southern ocean electric fish exist as dimers, in the same fashion as their northern hemisphere counterparts. The cDNAs of the receptor's four subunits were sequenced from Hypnos monopterigium and the northern hemisphere counterpart, Torpedo marmorata, while cDNAs from only two subunits, α and δ, were able to be sequenced from Narcine tasmaniensis. The penultimate amino acid in the δ subunit of each of the newly sequenced fish species is a cysteine residue. Its conservation suggests that the mechanism for the observed dimerization of acetylcholine receptors is disulfide bond formation between the δ subunit of adjacent receptors, analogous to acetylcholine receptor dimers observed in other electric fish. It appears that this mechanism for receptor clustering is unique to acetylcholine receptors packed and organized in the specialized organs of electric fish. Alignment of the deduced protein sequences with the equivalent sequences from Torpedo californica and humans reveals a high degree of homology.

End-Plate Acetylcholine Receptor: Structure, Mechanism, Pharmacology, and Disease

“…Electron microscopy of these tubes, embedded in amorphous ice, revealed that the unit AChR was an elongated cylinder with an extracellular projection of 65 Å, a transmembrane span of 30 Å, and an intracellular projection of 30 Å (177, 265). Each cylinder was joined to another identical structure to form a dimer through disulfide bonded cysteine residues at the COOH termini of the δ -subunits, a structural motif unique to electric rays (263). Evidence that the cylindrical structures were genuine AChRs included their selective location in postsynaptic membranes (117), their coincidence with α -neurotoxin binding (89), a similar size and shape to the purified AChR protein imaged by electron microscopy (27, 80, 143, 292), and functional reconstitution of purified AChRs in lipid bilayers (161).…”

“…The embryologic origin of the electric organ is the same as skeletal muscle, but the cells lack contractile filaments and form thin, flattened disks that stack in tall columns aligned side by side. Muscle AChRs are heteropentamers composed of α -, β -, and δ -subunits, plus either the fetal γ -subunit or the adult ε-subunit (257), although some animals, such as Torpedo , lack the ε-subunit (263). The subunits assemble in a sequence of oligomerization steps with intervening conformational changes (101) that minimize multiple or incorrect subunit stoichiometries, rendering assembly highly specific.…”

Rapid and simple protein-stability screens: application to membrane proteins

“…BtuCD, MscL and MscS from Escherichia coli were overexpressed and purified by members of D. C. Rees' laboratory as previously described (Chang et al, 1998;Bass et al, 2002;Locher et al, 2002). Acetylcholine receptor (AChR) was purified from the electric organ of Torpedo marmorata as previously described (Tierney et al, 2004). Protein concentrations were determined using the Pierce BCA assay.…”

In vivo adsorption of autoantibodies in myasthenia gravis using Nanodisc-incorporated acetylcholine receptor

“…The column was subsequently washed to remove any unincorporated AChR and ND-AChR particles were eluted. The eluted solution, containing a mixture of excess empty NDs and ND-AChR, was dialyzed against low-ionic strength Tris buffer (20 mM NaCl) and the solution was passed over TDAC affinity resin which contains an acetylcholine analog that binds only to intact and properly folded AChR (Tierney et al, 2004). Empty NDs were then removed by extensive washing in low-ionic strength buffer.…”

“…The column was washed with 50 mM imidazole and ND and ND-AChR particles were eluted with 0.4M imidazole. Ni-column eluate was dialyzed overnight at 4°C against low-ionic strength 20mM Tris-HCl buffer (pH 7.4), 20 mM NaCl and the solution was passed over an affinity resin comprised of the acetylcholine analog 4,7,10-trioxa-1,13-carboxyethyl-2-trimethylaminetridecanediaamine (TDAC) (Tierney et al, 2004). The column was extensively washed with 10 column volumes of low-salt buffer to eliminate empty NDs, and ND-AChR was eluted with a step gradient of 50–150 mM NaCl to enrich the sample for ND-AChR.…”