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Molecular genetics of catecholamines and manic depressive illness
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Diversity of γ-aminobutyrate type A (GABA_A) receptors has
recently been proposed to be achieved by assembly of receptor subtypes
from a multitude of subunits (α1-6, β1-3, γ1-2, and
δ) encoded by different genes. Here we report a further mechanism
for creating GABA_A receptor diversity: alternative RNA splicing. Two
forms of bovine γ2 subunit cDNA were isolated (γ2S and
γ2L) that differed by the presence or absence of a 24-base-pair
(8-amino acid) insertion in the cytoplasmic domain between the third and
fourth putative membrane-spanning regions. Polymerase chain reaction
from RNA demonstrated that the two forms of γ2 subunit are
expressed in bovine, human, and rat brain. Sequencing of genomic DNA
clones encoding the γ2 subunit demonstrated that the 24-base-pair
insert is organized as a separate exon. Analysis of the sequence of the
8-amino acid insert revealed that it contains a protein kinase C
consensus phosphorylation site. Expression of the large cytoplasmic loop
domains of γ2S and γ2L in Escherichia coli, followed by
phosphorylation of the recombinant proteins by protein kinase C,
demonstrated that γ2L, but not γ2S, could be phosphorylated.
Thus the two forms of γ2 subunit differ by the presence or absence
of a protein kinase C phosphorylation site. This mechanism for creating
GABA_A receptor diversity may allow differential regulation of the
function of receptor subtypes.
The murine chromosomal gene for the GABAA receptor δ subunit was isolated and characterized by high-resolution mapping and DNA sequencing. Spanning 13 kb, it comprises nine exons and displays an intron pattern comparable, but not identical, to that seen in members of the nicotinic acetylcholine receptor family. Notably, the second transmembrane domain thought to line the ion channel and conserved among different GABAA receptor subunits, is interrupted by an intron. The 5′-flanking region of the δ gene displays features characteristic of a CpG island and lacks canonical promoter elements such as TATA and CCAAT consensus sequences in proximity to the transcriptional initiation site. The human δ subunit gene was localized on the short arm of chromosome 1.
Hybridization of GABAA receptor probes to human chromosomes in situ and to DNA from sorted human chromosomes has localized the genes encoding a β subunit and three isoforms of the a subunit. The α2 and β genes are both located on chromosome 4 in bands p12–p13 and may be adjacent. The α1 gene is on chromosome 5 (bands q34–q35) and the α3 gene is on the X chromosome. The a3 locus was mapped also on the mouse X chromosome using genetic break-point analysis in an interspecies pedigree. The combined results locate the human a3 gene within band Xg28, in a location that makes it a candidate gene for the X-linked form of manic depression.
DNA sequences encoding two variants of a novel gamma-aminobutyric acidA (GABAA) receptor beta subunit were isolated from an embryonic chicken whole-brain cDNA library and a chicken genomic library. The coding regions of these variants only differ from each other by the absence or presence of 12 bp in the region that encodes the presumed intracellular loop between transmembrane domains M3 and M4; the encoded subunits have been named beta 4 and beta 4', respectively. The predicted mature polypeptides are 72-77% identical to the previously characterized mammalian and chicken beta 1, beta 2, and beta 3 subunits. Analysis of the beta 4-subunit gene reveals that the different transcripts encoding the two variants arise by the use of one of two 5'-donor splice sites that are separated by 12 bp. This is the first demonstration of alternative splicing of a GABAA receptor subunit gene transcript and represents a further mechanism for the generation of GABAA receptor heterogeneity.