Evolution of vertebrate nicotinic acetylcholine receptors

Abstract

Background: Many physiological processes are influenced by nicotinic acetylcholine receptors (nAChR), ranging from neuromuscular and parasympathetic signaling to modulation of the reward system and long-term memory. Due to the complexity of the nAChR family and variable evolutionary rates among its members, their evolution in vertebrates has been difficult to resolve. In order to understand how and when the nAChR genes arose, we have used a broad approach of analyses combining sequence-based phylogeny, chromosomal synteny and intron positions.

Results: Our analyses suggest that there were ten subunit genes present in the vertebrate predecessor. The two basal vertebrate tetraploidizations (1R and 2R) then expanded this set to 19 genes. Three of these have been lost in mammals, resulting in 16 members today. None of the ten ancestral genes have kept all four copies after 2R. Following 2R, two of the ancestral genes became triplicates, five of them became pairs, and three seem to have remained single genes. One triplet consists of CHRNA7, CHRNA8 and the previously undescribed CHRNA11, of which the two latter have been lost in mammals but are still present in lizards and ray-finned fishes. The other triplet consists of CHRNB2, CHRNB4 and CHRNB5, the latter of which has also been lost in mammals. In ray-finned fish the neuromuscular subunit gene CHRNB1 underwent a local gene duplication generating CHRNB1.2. The third tetraploidization in the predecessor of teleosts (3R) expanded the repertoire to a total of 31 genes, of which 27 remain in zebrafish. These evolutionary relationships are supported by the exon-intron organization of the genes.

Conclusion: The tetraploidizations explain all gene duplication events in vertebrates except two. This indicates that the genome doublings have had a substantial impact on the complexity of this gene family leading to a very large number of members that have existed for hundreds of millions of years.

Keywords: Acetylcholine; Gene duplication; Nicotinic; Ohnolog; Paralogon; Receptor; Spotted gar; Synteny; Tetraploidization; Zebrafish.

Fig. 1

Phylogenetic maximum likelihood tree of the nAChR genes, rooted with the human 5HTR3A and 5HTR3B; the root is not displayed. The collapsed nodes represent the orthologs of the respective nAChR genes, where each color code corresponds to one nAChR gene subfamily. The invertebrate chordate (represented by amphioxus and tunicates) and protostome (represented by C. elegans and fruitfly) closest homologs are shown as non-colored nodes. The tree topology is supported by a non-parametric Ultra-Fast Bootstrap (UFBoot) analysis

Fig. 2

The left panel shows a phylogenetic tree based on intron insertions into the vertebrate sequences. The top panel shows a schematic outline of the general protein domain structure, including the extracellular domain (ECD), the binding domain (BD), the transmembrane regions 1–3 (TM1-TM3), the large intracellular domain (ICD) and finally the last TM4 region and the short extracellular carboxy-terminus. The Cys-loop with its conserved proline is marked in yellow. Below follows the exon-intron organization for each gene, if the organization is the same in two or more genes only one of them is shown. The color coding of the exons corresponds to the schematic outline in the top panel. The exons are drawn to scale and the splice phase is indicated at the beginning of each intron. Common intron positions are marked with a line, combining the positions that are identical between the different genes. The introns are numbered (1–20). The curved line indicates a common position shared between the CHRNB1/CHRNB1.2 genes and the CHRNA1 gene. Positions of cysteines, N-linked glycosylation sites (those with the same color are in corresponding positions) and the cysteine pair are also marked for each gene. Faded color means that it is not present in all orthologs

Fig. 3

Analysis of the evolutionary history of the nAChR family with chromosomal locations of the nAChR genes and their neighboring gene families in human, chicken and spotted gar. Crosses indicate gene loss or gene not yet identified. Chicken and spotted gar illustrations are re-used with permission from Daniel Ocampo Daza, source: www.egosumdaniel.se and the human image is re-used with permission from https://commons.wikimedia.org. The duplication scheme and neighboring gene families for the CHRNA6/CHRNA3, CHRNA11/CHRNA8/CHRNA7, CHRNB2/CHRNB5/CHRNB4 and CHRNB3/CHRNA5 genes. The right panel summarizes chromosomes included. This paralogon is referred to as paralogon 1 in this study

Fig. 4

Analysis of the evolutionary history of the nAChR family with chromosomal locations of the nAChR genes and their neighboring gene families in human, chicken and spotted gar. Crosses indicate gene loss or gene not yet identified. Chicken and spotted gar illustrations are re-used with permission from Daniel Ocampo Daza, source: www.egosumdaniel.se and the human image is re-used with permission from https://commons.wikimedia.org. The duplication scheme and neighboring gene families for the CHRNA2/CHRNA4 genes. This region of gene families have been analyzed in depth in previous studies from our lab, see Dreborg et al. (2008) and Cardoso et al. (2016). The right panel summarizes chromosomes included. This paralogon is referred to as paralogon 2 in this study

Fig. 5

Analysis of the evolutionary history of the nAChR family with chromosomal locations of the nAChR genes and their neighboring gene families in human, chicken and spotted gar. Crosses indicate gene loss or gene not yet identified. Chicken and spotted gar illustrations are re-used with permission from Daniel Ocampo Daza, source: www.egosumdaniel.se and the human image is re-used with permission from https://commons.wikimedia.org. The duplication scheme and neighboring gene families for the CHRNA9/CHRNA10 genes. The right panel summarizes chromosomes included. This paralogon is referred to as paralogon 3 in this study.

Fig. 6

Analysis of the evolutionary history of the nAChR family with chromosomal locations of the nAChR genes and their neighboring gene families in human, chicken and spotted gar. Crosses indicate gene loss or gene not yet identified. Chicken and spotted gar illustrations are re-used with permission from Daniel Ocampo Daza, source: www.egosumdaniel.se and the human image is re-used with permission from https://commons.wikimedia.org. The duplication scheme and neighboring gene families for the CHRNA1 gene. This region has been analyzed in detail in previous studies from our lab, see Larhammar et al. (2002), Sundström et al. (2008) and Widmark et al. (2011). This paralogon is referred to as paralogon 4 in this study

Fig. 7

Analysis of the evolutionary history of the nAChR family with chromosomal locations of the nAChR genes and their neighboring gene families in human, chicken and spotted gar. Crosses indicate gene loss or gene not yet identified. Chicken and spotted gar illustrations are re-used with permission from Daniel Ocampo Daza, source: www.egosumdaniel.se and the human image is re-used with permission from https://commons.wikimedia.org. The neighboring gene families and duplication schemes for the CHRNB1, CHRND and CHRNE/CHRNG genes. The spotted gar retained a local duplication of the CHRNB1 gene, forming CHRNB1.2. The right panel summarizes chromosomes included. This paralogon is referred to as paralogon 5 in this study

Fig. 8

Analysis of the evolutionary history of the nAChR family with chromosomal location of the nAChR genes and their neighboring gene families in zebrafish, medaka, stickleback and fugu. Crosses indicate gene loss or gene not yet identified. Species illustrations are re-used with permission from Daniel Ocampo Daza, source: www.egosumdaniel.se

Fig. 9

a Duplication scheme of the nAChR genes following 1R + 2R. Ten nAChR genes in five different paralogons are present in the vertebrate predecessor. Following 1R + 2R the repertoire expands to 19 genes. Human retained 16 genes (lacking CHRNA11, CHRNA8 and CHRNB5), chicken 15 (lacking CHRNA11, CHRNB5, CHRNB1 and CHRNE) and spotted gar 18 (lacking CHRNB2), plus the CHRNB1.2 local duplication. b Duplication scheme of the nAChR genes following 3R. 20 nAChR genes are present in the teleost predecessor, following 3R the repertoire expands to 31 genes. Zebrafish has retained 27 genes. Crosses indicate gene loss or gene not yet identified