Phylogenetic conservation of disulfide-linked, dimeric acetylcholine receptor pentamers in southern ocean electric rays.
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 alpha-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, alpha and delta, were able to be sequenced from Narcine tasmaniensis. The penultimate amino acid in the delta 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 delta 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.
Tierney
M L
ML
School of Biochemistry & Molecular Biology, Australian National University, Canberra, ACT 0200, Australia. louise.tierney@anu.edu.au
Osborn
K E
KE
Milburn
P J
PJ
Stowell
M H B
MH
Howitt
S M
SM
D000818:Animals; D001315:Australia; D001483:Base Sequence; D002855:Chromatography, Thin Layer; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D019143:Evolution, Molecular; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009792:Oceans and Seas; D010802:Phylogeny; D011485:Protein Binding; D011950:Receptors, Cholinergic / Q000737:chemistry* / Q000235:genetics* / Q000648:ultrastructure; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017385:Sequence Homology; D014101:Torpedo / Q000235:genetics*
D017931:DNA Primers; D018076:DNA, Complementary; D011950:Receptors, Cholinergic