Evolution of GABA_A receptor diversity in the human genome

Abstract

Nowhere is the record of receptor evolution more accessible than in the organization of the 19 vertebrate genes coding for subunits of the major inhibitory neurotransmitter receptor in the central nervous system, the γ-aminobutyric acid receptor (GABA_AR). Co-expression of α, β, and γ subunit genes is necessary for the formation of a GABA_AR that is potentiated by widely used anxiolytics, anticonvulsants, and hypnotics. The identification of α, β, and γ genes on chromosomes 4, 5, and 15 suggests that co-localization of a γ gene with an α and β may be important for brain function. We have now directly examined the organization of GABA_AR subunit genes on human chromosomes. Estimates of physical distance using in situ hybridization to cells in interphase, and gene localization using hybridization to cells in metaphase demonstrate the existence of β–α–α–γ gene clusters in cytogenetic bands on chromosomes 4(p12) and 5(q34). Sequencing of PAC clones establishes intercluster conservation of a unique head-to-head configuration for α and β genes on chromosomes 4, 5, and 15. Remarkably, phylogenetic tree analysis predicts the existence of a β–α–γ ancestral gene cluster in which internal duplication of an ancestral α was followed by cluster duplication, resulting in the relative chromosomal positions of modern GABA_AR subunit genes in the human genome.

Introduction

Neurotransmitter receptors convert chemical into electrical signals at the postsynaptic membranes of classical synapses. The discovery that ligand-gated ion channels, the most prevalent form of neurotransmitter receptor, are assembled from a multiplicity of homologous subunits encoded by independent genes has raised a major question as to the evolution of the nervous system: How did such an extensive diversity of receptor subunits evolve? With at least 19 different vertebrate genes coding for receptor subunits (for review see Rabow et al., 1995; Levin et al., 1996; Wilke et al., 1997), the pentameric (Nayeem et al., 1994) γ-aminobutyric acid receptor (GABA_AR) is an exquisite example of how gene duplication can provide the foundation for the expression of related receptor subunits, each with its own unique developmental and tissue-specific distribution (Laurie et al., 1992; Wisden et al., 1992).

Our previous localization of the β2 subunit gene to 5q34-35 that identified a putative α–β–γ gene cluster for the major isoform of the GABA_AR when taken together with conservation of intron position in the β1, β2, β3, and β4 genes led us to propose that the majority of GABA_A receptor subunit genes arose from the duplication and subsequent translocation of an ancestral gene cluster (Russek and Farb, 1994). Additional putative α–β–γ clusters have been identified on chromosomes 4 (Buckle et al., 1989; Wilcox et al., 1992; McLean et al., 1995) and 15 (Knoll et al., 1993; Sinnet et al., 1993). A new subunit gene class termed ε (Davies et al., 1997; Wilke et al., 1997) has been mapped to a region on the X chromosome that contains the α3 gene and a putative β4-like subunit gene (Levin et al., 1996), suggesting that there are at least four related clusters in the human genome. Moreover, the fact that there are two alpha genes, one beta, and one gamma on chromosomes 4 (McLean et al., 1995) and 5 (Johnson et al., 1992; Hicks et al., 1994) indicates that simple gene duplication cannot account for the expansion in GABA_AR gene number.

We have now directly examined the cluster organization of GABA_AR subunit genes by fluorescence in situ hybridization (FISH) interphase mapping using PAC clones that contain fragments of GABA_AR subunit genes. Conservation of gene order, intergenic distance, and unique head-to-head configuration for the transcriptional units of α and β genes on chromosomes 4, 5, and 15 demonstrate the occurrence of cluster duplication and translocation. Results of phylogenetic tree analysis also predict duplication of an ancestral cluster in which internal duplication of an alpha gene expanded the diversity of the α subunit GABA_A receptor gene family.