Identification of three novel Ca(2+) channel gamma subunit genes reveals molecular diversification by tandem and chromosome duplication

Abstract

Gene duplication is believed to be an important evolutionary mechanism for generating functional diversity within genomes. The accumulated products of ancient duplication events can be readily observed among the genes encoding voltage-dependent Ca(2+) ion channels. Ten paralogous genes have been identified that encode isoforms of the alpha(1) subunit, four that encode beta subunits, and three that encode alpha(2)delta subunits. Until recently, only a single gene encoding a muscle-specific isoform of the Ca(2+) channel gamma subunit (CACNG1) was known. Expression of a distantly related gene in the brain was subsequently demonstrated upon isolation of the Cacng2 gene, which is mutated in the mouse neurological mutant stargazer (stg). In this study, we sought to identify additional genes that encoded gamma subunits. Because gene duplication often generates paralogs that remain in close syntenic proximity (tandem duplication) or are copied onto related daughter chromosomes (chromosome or whole-genome duplication), we hypothesized that the known positions of CACNG1 and CACNG2 could be used to predict the likely locations of additional gamma subunit genes. Low-stringency genomic sequence analysis of targeted regions led to the identification of three novel Ca(2+) channel gamma subunit genes, CACNG3, CACNG4, and CACNG5, on chromosomes 16 and 17. These results demonstrate the value of genome evolution models for the identification of distantly related members of gene families.

Figure 1

(A) Amino acid alignment of the voltage-dependent Ca²⁺ channel γ_1–5 subunits. The relative positions of the three introns are indicated by dots, and the adjacent exons are numbered 1–4. The location of putative transmembrane domains predicted by the program TMpred (Hofmann and Stoffel 1993) are underlined (see Fig. 2). Dashes indicate gaps introduced to maintain optimal alignment. HG1, 2, 3, 4, and 5 were translated from sequences of the human genes CACNG1–CACNG5, respectively, with conceptual splicing at positionally conserved splice sites. Consensus N-glycosylation sites are double-underlined. Potential phosphorylation sites, indicated by carets (^), are consensus targets for one or more of the following: cAMP/cGMP-dependent protein kinase (Prosite PDOC00004), protein kinase C (PDOC00005), casein kinase II (PDOC00006), and tyrosine kinase (PDOC00007). Potential protein kinase C phosphorylation sites at amino acids 50 and 51 of HG2 are not marked. A single nontransmembrane region of well-conserved amino acid sequence is indicated by diamonds (♦). (B) Comparison of the intron–exon splice junctions and intron sizes of the human CACNG1–5 genes (HG1–5). Exon sequences are in uppercase letters; intron sequence in lowercase. Consensus splice acceptor and donor motifs (Mount 1982) are indicated at the bottom. (C) Alignment of CACNG1–5 translation initiation sites. The consensus translation initiation motif (Kozak 1984) is shown at the bottom. The putative first methionine codon is underlined. The sequences of the CACNG2 and CACNG3 genes are identical for 9 nucleotides preceding the start codon; however, this sequence is a poor match to the consensus (thymine is the most uncommon residue at positions −1 and −2 of vertebrate translation start sites, 9% and 11%, respectively). (M) A or C; (R) purine; (Y) pyrimidine; (N) any base.

Figure 2

Comparison of transmembrane topologies of Ca²⁺ channelγ_1–5 subunits predicted by the TMpred program (Hofmann and Stoffel 1993). Positive TMpred values (y-axis, ×1000) indicate likely membrane spanning segments. Amino acid position is shown on the x-axis. All five γ subunit isoforms are predicted to contain four transmembrane domains with amino and carboxyl termini located intracellularly.

Figure 3

(A) Molecular phylogeny of the Ca²⁺ channel γ subunit family. The alignment used to infer the tree was done independently for full-length proteins and for conserved regions alone (defined as translation start to the final residue of the fourth predicted transmembrane domain), and resulted in identical topologies. The number of trees with a particular node among 10,000 bootstrap replicates is indicated at the node (values for conserved domain trees shown above the horizontal line and for full length proteins below). A member of the related Claudin protein family, Claudin 4, was included in the comparison and defined as the out-group. Branch lengths are arbitrary and do not correspond to genetic distances. This tree is unrooted. (B) Pair-wise amino acid identity among the Ca²⁺ channel γ_1–5 subunits and mouse Claudin 4. Percent identity determined after alignment by the BLAST2 program is shown above the horizontal and by the ALIGN program below the horizontal. Homology between Claudin 4 and the γ subunits was below the threshold of detection by BLAST2. (MC4) Mouse Claudin 4; HG1, 3, 4, and 5 refer to human Ca²⁺ channel γ subunits; MG2 refers to the mouse γ₂ subunit; (n.a.), not applicable.

Figure 4

A model of Ca²⁺ channel γ subunit gene family evolution. Integration of chromosome locations and molecular phylogeny data suggest a minimum of two tandem duplications of an ancestral γ subunit gene, followed by two duplications of the precursor of modern chromosome bands 16p11–p13, 17q11–q25, and 22q11–q24. Four potential genes suggested by the model (C′, C′′, A′, and A′′) but not identified in this study may have been lost during evolution sometime after the duplication indicated by arrow 3. Arrows 1 and 2 indicate tandem gene duplication events. Arrows 3 and 5 indicate regional (chromosome or whole genome) duplications. Letters (A, B, C) and symbols (′ , ′′) designate inferred ancestors of modern γ subunit genes.

Figure 5

(A–C) Physical maps of the region surrounding the human Ca²⁺ channel γ subunit genes. Genomic sequences are indicated by thin lines below the name of the associated clone, with selected markers shown below. (●) Sequence overlap; (○) end sequences associated only with a single parent clone in GenBank. Characterized genes are represented by thick lines, with transcriptional orientation indicated by arrowheads. PRKCB1 is interrupted by a gap in the genomic sequence of unknown length. Only the 3′ ends of PRKCA and ORFA05 are currently represented in GenBank. (CBP–P22) calcineurin-related gene; (PRKCB1) protein kinase C β1; (CACNG3) calcium channel γ3; CSF2RB colony-stimulating factor 2 receptor β; (NCF4) neutrophil cytosolic factor 4; (PVALB) parvalbumin; (HSRASR) similar to H. sapiens RAY1 gene; (CACNG2) calcium channel γ2; (HPSP1) Hermansky–Pudlak syndrome pseudogene; (EIF3–P66) eukaryotic translation initiation factor 3, subunit 7 (ζ, 66/67 kD); (TRX2) thioredoxin 2; (PRKCA) protein kinase C α; (G6PDP1) formerly G6PDL, glucose-6-phosphate dehydrogenase pseudogene 1; (CACNG5) calcium channel γ5; (CACNG4) calcium channel γ4; (CACNG1), calcium channel γ1; (ORFA05) hypothetical myeloid cell line protein 5. StsG25737 represents an EST of CACNG3. Complete clone names and database accession numbers are given in Methods. (D–F) Comparison of the intron–exon structure of CACNG1–5. The scale bar in D applies to D–F.