Gene Structure and Transcriptional Regulation of the Neuronal Nicotinic Acetylcholine Receptors

Abstract

The use of recombinant DNA techniques to advance the biology of nicotinic acetylcholine receptors (nAChRs) began in 1980, after the seminal demonstration by microsequencing that the electroplax nAChR of _Torpedo consisted of four homologous subunits (RAFTERY et al. 1980). Prompted by this finding, several research groups independently decided that the tools were available to clone the nAChR cDNAs. The electric organ was readily obtainable and it was a good source of receptor mRNA that could be translated in vitro to synthesize labeled receptor subunits. The subunits could then be formally identified by immunoprecipitation using antireceptor antibodies. In one approach, pools of electric organ cDNA clones were denatured and bound to a solid matrix. Passing electric organ mRNA on the matrix in hybridizing conditions subtracted the corresponding messengers, which were eluted and translated in vitro. Pools were scored as positive if the mRNAs they bound drove the synthesis of immunoprecipitable protein. They were divided up until single clones capable of retaining receptor mRNA were isolated (BALLIVET et al. 1982). In short order, groups led by Barnard, Changeux, Heinemann, and Numa reported the isolation and sequence of the cDNAs encoding the electric organ nAChR, thereby effecting the first complete cloning of a multi-subunit ligand- gated ion channel. The four conserved hydrophobic domains and two long hydrophilic domains of the subunits immediately suggested an insertional topology that is still accepted today (SUMIKAWA et al. 1982; DEVILLERS-THIERY et al. 1983; CLAUDIO et al. 1983; NODA et al. 1982). Availability of the Torpedo subunits led to the rapid isolation of their homologues in muscle cDNA libraries from rat, bovine, chicken, human, mouse, and other vertebrate species. The very high degree of conservation of the muscle nAChR subunits throughout vertebrate space argued that this set of genes must predate the vertebrate radiation. Indeed, additional cloning work soon established that bona fide nAChR genes are found in invertebrates, including _Drosophila where they must have a neural function since the neuromuscular junction of insects is not cholinergic (BOSSY et al. 1988; HERMANS-BORGMEYER et al. 1989). Meanwhile, abundant evidence was being obtained in vertebrates that neuronal nAChRs were closely related to their muscle counterparts in sequence, structure, and function (BOULTER et al. 1986; NEF et al. 1988). The detailed study of their physiology was made possible by the development of a convenient functional assay. Upon being injected with electric organ mRNA, _Xenopus oocytes assembled fully functional _Torpedo receptors in their plasma membrane, and these could easily be studied by standard physiological and pharmacological procedures (BARNARD et al. 1982). The assay was quickly adapted to the expression of cloned muscle (SAKMANN et al. 1985) and neuronal (BOULTER et al. 1987; BALLIVET et al. 1988) nAChR subunits. Enormous advances in the field have been derived from this meeting of molecular biology and electrophysiology in the confines of the _Xenopus oocyte. Not only could naturally occurring combinations of subunits be tested for function, but point mutants, deletion mutants, hybrids, and chimeras could also be assayed (IMOTO et al. 1988; COOPER et al. 1991; GROSS et al. 1991; GALZI et al. 1992). In this respect, the role of the neuronal α7 subunit cannot be overemphasized. Because it readily assembles as a homomeric channel in oocytes (COUTURIER et al. 1990a), the α7 receptor has become the most widely used model system in nAChR structure-function studies (REVAH et al. 1991; DEVILLERS-THIERY et al. 1993). As useful and convenient as it is, the _Xenopus system has drawbacks: it consumes animals at a high rate and the oocytes require delicate preliminary treatment in order to make clean plasma membrane accessible to the patch pipette. Systems such as the internodal cells of the alga _{Chara corallina} are being explored (LUHRING and WITZEMANN 1995) and may some day provide cheaper and more humane alternatives.