A single gene codes for the nicotinic acetylcholine receptor alpha-subunit in Torpedo marmorata: structural and developmental implications.

Abstract

We have used Southern blot hybridization to analyze the genomic structure encoding the alpha‐subunit of the acetylcholine receptor (AChR) in Torpedo marmorata, with cDNA probes isolated from the electric organ. Four different radiolabelled probes, corresponding to various parts of the alpha‐subunit mRNA, hybridized to several genomic fragments of T. marmorata DNA generated by digestion with the restriction enzymes SstI, PvuII and PstI. The same hybridization pattern was observed after washing the blots under low‐ or high‐stringency conditions. As a check for detection sensitivity of heterologous sequences, the same probes were hybridized to PvuII‐digested chicken DNA, revealing bands at low stringency which disappeared at higher stringencies. Unambiguously, two of our probes (one of them entirely within the coding region) hybridized to a single genomic fragment from T. marmorata DNA. This feature, as well as the results of an extensive study of the whole hybridization pattern, points towards the uniqueness of alpha‐subunit‐specific sequences in the genome of T. marmorata. Since overall more bands were found than expected from the cDNA sequence, this alpha‐subunit gene must be split by several introns (at least four, possibly more). The length of this gene is at least 20 kb. The existence of a single alpha‐subunit gene is consistent with the absence of chemical heterogeneity in the NH2‐terminal sequence of the purified alpha‐chain, and supports the view that the two alpha‐chains belonging to one AChR oligomer have an identical primary structure. It also suggests that localization and stabilization of the AChR in well‐defined post‐synaptic areas of T. marmorata electric organ basically relies, during development, on ‘epigenetic’ mechanisms.

Activity regulates the levels of acetylcholine receptor alpha-subunit mRNA in cultured chicken myotubes.

“…The number of isolated clones (six in four to six genomic equivalents) is also consistent with a single-copy gene. We conclude that, within our detection limits, the a-subunit gene is encoded by a unique locus in chicken, as was found for T. marmorata (26) and mouse (31). Gene families could be suggested to account for the occurrence of a-bungarotoxin-binding sites and nicotinic AcChoR in the central nervous system of vertebrates (34), particularly in the optic lobe of the chicken (35), for developmental changes in several properties of the AcChoR (36), and for differences between the two agonist binding sites, carried mainly by the two a subunits present per pentameric AcChoR (1).…”

“…Dideoxy sequencing (21) (600 Ci/mmol, Amersham) as the label. M13 subclones also were used to obtain single-stranded uniformly 32P-labeled probes (22) (26). Final washes of the RNA and DNA blots were routinely done in 30 mM NaCl/3 mM sodium citrate/0.1% NaDodSO4 at 450C (low stringency).…”

Functional architecture of the nicotinic acetylcholine receptor: A prototype of ligand-gated ion channels

“…The two a subunits of the Torpedo marmorata receptor (Klarsfeld et al, 1984) and that of mouse muscle nAChR (Merlie et al, 1983) are encoded by a single gene, but the two agonist binding sites are not strictly equivalent. They differ by their kinetics of binding and dissociation of a bungarotoxin (Weber & Changeux 1974a, b, c;Maelicke & Reich, 1976;Maelicke et al, 1977;Kang & Maelicke, 1980;Maelicke et al, 1989), by their affinities for the competitive antagonists d-tubocurarine (Neubig & Cohen, 1979;Pedersen & Cohen, 1990a) or decamethonium and by their different labeling sensitivity to affinity reagents such as bromoacetylcholine, MBTA (Karlin et al, 1976;Damle & Karlin, 1978;Delegeane & McNamee, 1980;Ratnam et al, 1986) or lophotoxin (Culver, Fenical & Taylor, 1984).…”

Structure linkage, and sequence of the two genes encoding the delta and gamma subunits of the nicotinic acetylcholine receptor.

“…Recent reports (25,28) have established that the 8 and a subunit genes are unique in the Torpedo genome.…”