Ralf Schoepfer

Brain α-bungarotoxin binding protein cDNAs and MAbs reveal subtypes of this branch of the ligand-gated ion channel gene superfamily

alpha-Bungarotoxin (alpha Bgt) is a potent, high-affinity antagonist for nicotinic acetylcholine receptors (AChRs) from muscle, but not for AChRs from neurons. Both muscle and neuronal AChRs are thought to be formed from multiple homologous subunits aligned around a central cation channel whose opening is regulated by ACh binding. In contrast, the exact structure and function of high-affinity alpha Bgt binding proteins (alpha BgtBPs) found in avian and mammalian neurons remain unknown. Here we show that cDNA clones encoding alpha BgtBP alpha 1 and alpha 2 subunits define alpha BgtBPs as members of a gene family within the ligand-gated ion channel gene superfamily, but distinct from the gene families of AChRs from muscles and nerves. Subunit-specific monoclonal antibodies raised against bacterially expressed alpha BgtBP alpha 1 and alpha 2 subunit fragments reveal the existence of at least two different alpha BgtBP subtypes in embryonic day 18 chicken brains. More than 75% of all alpha BgtBPs have the alpha 1 subunit, but no alpha 2 subunit, and a minor alpha BgtBP subtype (approximately 15%) has both the alpha 1 and alpha 2 subunits.

cDNA clones coding for the structural subunit of a chicken brain nicotinic acetylcholine receptor

Nicotinic acetylcholine receptors (AChRs) immunoaffinity-purified from brains are composed of only two kinds of subunits rather than the four kinds present in muscle-type AChRs. Here we report the N-terminal protein sequences of the structural subunits of AChRs from rat and chicken brains and the cloning of full-length cDNAs for the chicken brain AChR structural subunit. Previously, the N-terminal amino acid sequence of the ACh-binding subunit of AChR immunoaffinity-purified from rat brain was shown to correspond to the cDNA alpha 4. Thus, cDNA sequences are now known for both of the subunits that form one AChR subtype in vivo.

EXPRESSION OF NICOTINIC ACETYLCHOLINE-RECEPTOR SUBTYPES IN BRAIN AND RETINA

Neuronal nicotinic acetylcholine receptors (AChRs) are composed of two types of subunits: ACh-binding (termed alpha 2, alpha 3, alpha 4 ...) and structural (termed beta 2, beta 3, beta 4 ...). AChR subtypes composed of combinations of subunits of these two types encoded by several related genes are expressed in different parts of the nervous system, where they presumably serve different functional roles. Here we identify the ACh-binding subunit of the most prominent chicken brain AChR subtype by N-terminal amino acid sequence and show that it corresponds to the alpha 4 gene. Previously we identified the structural subunit for this AChR subtype from chicken brain as beta 2 by N-terminal amino acid sequence. Thus, this identifies both genes which encode subunits of the major nicotinic AChR subtype in avian brains. By immunoprecipitation, immunohistochemistry, and northern blot analysis we show that alpha 3 (or a very closely related sequence) is expressed at low levels in the brain and relatively high levels in the retina, while alpha 4 is expressed at high levels in the brain and lower levels in the retina. This differential expression indicates that alpha 3-containing ganglionic-type AChRs may be an important AChR subtype in avian retina.

The pRSET family of T7 promoter expression vectors for Escherichia coli

A family of eight T7 promoter-based expression plasmids is presented. These are high-copy-number vectors featuring translational start and stop elements and a multiple cloning site (polylinker) with eleven unique restriction sites in all six reading frames. Depending on the cloning strategy used, recombinant proteins may contain either short vector-encoded fusion fragments or no fusion fragments at all. Following promoter induction, proteins are usually produced at a high level.

The human medulloblastoma cell line TE671 expresses a muscle-like acetylcholine receptor Cloning of the α-subunit cDNA

Nicotinic acetylcholine receptors (AChRs) from muscle bind α‐bungarotoxin (αBgt) and are composed of four kinds of subunits, whereas AChRs from mammalian brains do not bind αBgt and are composed of two kinds of subunits. αBgt‐binding proteins whose function is unknown are also found in brain. All these proteins belong to the same gene family. The human medulloblastoma cell line TE671 expresses a functional AChR which binds αBgt. Surprisingly, the AChR of this neuron‐derived cell line has electrophysiological, immunological and biochemical properties different from neuronal AChRs and very similar to muscle AChRs. The TE671 AChR binds αBgt, but is different from αBgt‐binding proteins in brain. Here we show that TE671 expresses the α‐subunit mRNA coding for the muscle AChR, thereby proving that TE671 expresses a muscle‐type AChR that is not expressed in adult brain. The isolated cDNA clones should prove useful for expression of large amounts of human muscle‐type AChR α‐subunit protein for studies of the autoimmune response to muscle AChRs in human myasthenia gravis.

Antisera against an acetylcholine receptor α3 fusion protein bind to ganglionic but not to brain nicotinic acetylcholine receptors

Neuronal nicotinic acetylcholine receptor (AChR) subtypes have been defined pharmacologically, immunologically, and by DNA cloning, but the correlations between these approaches are incomplete. Vertebrate neuronal AChRs that have been isolated are composed of structural subunits and ACh‐binding subunits. A single kind of subunit can be used in more than one AChR subtype. Monoclonal antibody (mAb) 35 binds to structural subunits of subtypes of AChRs from both chicken brain and ganglia. By using antisera to a unique sequence of α3 ACh‐binding subunits expressed in bacteria, we show that ganglionic AChRs contain α3 ACh‐binding subunits, whereas the brain AChR subtype that binds mAb 35 does not. Subunit‐specific antisera raised against recombinant proteins should be a valuable approach for identifying the subunit composition of receptors in multigene, multisubunit families.

The nicotinic acetylcholine receptor gene family: structure of nicotinic receptors from muscle and neurons and neuronal alpha-bungarotoxin-binding proteins.

Three branches of the ligand-gated ion channel gene superfamily encode proteins that bind cholinergic ligands: 1) nicotinic acetylcholine receptors (AChRs) from skeletal muscle, 2) nicotinic AChRs from neurons, and 3) α-bungarotoxin-binding proteins (αBgtBPs) from neurons. AChRs from vertebrate muscles and nerves differ in subunit composition, and in some cases in functional role, but both appear to be formed from several homologous subunits which form ACh-gated cation channels. αBgtBPs from vertebrate neurons have uncertain subunit compositions, uncertain endogenous ligands, and unknown functions. The ligand-gated ion channel gene superfamily also includes receptors for GABA and glycine, which are ligand-gated anion channels, and it probably also includes other ligand-gated ion channels (Barnard et al., 1987; Betz and Becker, 1988). The relation of glutamate receptors to this superfamily is less certain (Gregor et al., 1989; Hollmann et al., 1989; Wada et al., 1989).

Identification of a single melanin-concentrating hormone messenger ribonucleic acid in Coho Salmon : structural relatedness with 7SL ribonucleic acid

Melanin‐concentrating hormone (MCH) is a cyclic neuropeptide possessing antagonistic function to α‐melanocyte‐stimulating hormone and corticotropin‐releasing factor in the control of melanosome dispersion within melanophores and adrenocorticotropin release in fish. We have isolated and characterized MCH cDNAs from coho salmon (Oncorhyncus kisutch). The precursor protein predicted by the longest cDNA consists of 132 amino‐acids with a characteristic signal peptide at the N‐terminus and the biologically active salmon MCH (sMCH) peptide at the C‐terminus. The coho sMCH mRNA and protein sequences are very similar but not identical to the previously reported chum or chinook salmon counterparts, suggesting the existence of species polymorphism. Sequence similarities were revealed between α‐melanocyte‐stimulating hormone and part of the C‐terminal domain of sMCH precursor. Two sMCH genes were found in coho salmon. By contrast to other salmon species, only one major sMCH mRNA was detected in coho species suggesting that differential MCH gene expression might occur in salmon. In addition, under low stringency oligoprobes complementary to the sMCH RNA recognize a 0.3 kb RNA which was identified as the 7SL RNA. The regions conserved between those RNAs fold in a similar secondary structure. These similarities might reflect common ancestry which may have functional significance.

Structure of Neuronal Nicotinic Receptors

Nicotinic acetylcholine receptors (AChRs) from muscles and nerves form a gene family which is part of a gene superfamily of ligand-gated ion channels formed from multiple homologous subunits (reviewed in Lindstrom et al., 1987b; Barnard et al., 1987). The muscle-like AChRs from Torpedo electric organ are the best characterized protein in this family. Using monoclonal antibody (mAb) and cDNA probes initially derived from studies of electric organ AChRs, it has recently been possible to characterize neuronal AChRs. Although neuronal AChRs exhibit sequence homologies with muscle AChRs that reveal their evolution from common precursors, these AChRs differ in sub-unit structure, pharmacological properties, and functional roles.

Structure of Muscle and Neuronal Nicotinic Acetylcholine Receptors

Nicotinic acetylcholine receptors (AChRs) of muscle, brain, and ganglia belong to the ligand-gated cation channel family, whose most well-studied member is the AChR of the electric organs of Torpedo californica and related species. The strychnine-binding glycine receptor of the spinal cord and the brain γ-aminobutyric acidA (GABAA) receptor are ligand-gated anion channels which share sequence homologies with AChRs, which suggest that they are part of a ligand-gated receptor superfamily with a common evolutionary origin (Lindstrom et al., 1987b; Barnard et al., 1987). The neuronal α-bungarotoxin- (α-Bgt) binding protein, whose function and exact structure is still elusive, is probably a member of this superfamily, and surely more members will be discovered. Muscarinic AChRs (Peralta et al., 1988), on the other hand, belong to a completely different superfamily of receptors, with rhodopsin (Nathans & Hogness, 1983), adrenergic (Dixon et al., 1986), and serotonin receptors as members, and mediate their action through coupling proteins (G-proteins) (Kerlavage, 1987; Julius et al., 1988).