Genomic organization of the mouse and human ␣2␦2
voltage-dependent calcium channel subunit genes
Jane Barclay,* Michele Rees
Department of Paediatrics and Child Health, Royal Free and University College Medical School, The Rayne Institute, University College London,
5 University Street, London, WC1E 6JJ, UK
Received: 8 May 2000 / Accepted: 25 July 2000
Changes in intracellular Ca
concentration regulate a wide variety
of cellular function (Berridge et al. 1998). Voltage-dependent Ca
channels (VDCCs) are found in the plasma membranes of all ex-
citable cells and are hetero-oligomeric complexes composed of up
to five subunits: ␣1, ␣2, ␦, ␤ and ␥. The ␣1 subunit acts as a
channel pore, voltage sensor, and receptor for many drugs. The
other accessory subunits modify channel properties such as current
magnitude and kinetics (Walker and De Waard 1998).
The ␣2 and ␦ chains are derived by proteolytic cleavage of the
same gene product, the ␦ subunit acting as a membrane anchor for
the extracellular ␣2 subunit. Three genes encoding ␣2␦ subunits
have been described: human, mouse, rat, and rabbit CACNA2D1
(␣2␦1), human CACNA2D2 (␣2␦2), and mouse Cacna2d3 (␣2␦3)
(Klugbauer et al. 1999).
Genes encoding VDCCs have been implicated in the aetiology
of a wide range of mammalian phenotypes (Fletcher et al. 1998).
The genes encoding the ␣1A, ␤4, and ␥2 subunits have been
shown to underlie the mouse epilepsy phenotypes tottering, lethar-
gic, and stargazer (Fletcher and Frankel 1999). Recently, we have
demonstrated that the Cacna2d2 gene is also associated with a
phenotype of ataxia and epilepsy in ducky mice (unpublished).
The Cacna2d2 gene is located on mouse Chromosome (Chr) 9
approximately 59–60 cM from the centromere, and the human
ortholog CACNA2D2 is located on human Chr 3p21.3 (Gao et al.
Here, the genomic structures of the mouse Cacna2d2 and hu-
man CACNA2D2 genes are described and compared for the first
time. The two regions of alternative splicing identified in mouse
brain RNA are discussed together with a consideration of the as-
sociated non-consensus splice sites.
The 5.5-kb human CACNA2D2 cDNA (GenBank AF042792)
sequence has been previously described, and we have isolated
the mouse Cacna2d2 cDNA sequence (GenBank AF247139). Ge-
nomic clones containing the Cacna2d2 gene were identified from
the WI/MIT YAC library (y203E7, y257D12, y465F1) and the
RPCI21 mouse PAC library (p428C5, p432G2, p524O8, and
p524G24). Most of the intron/exon boundaries were determined by
using the Expand™ Long Template PCR system (Roche Diagnos-
tics, UK) to amplify mouse genomic and YAC DNA with primers
contained within the cDNA sequence. PCR products were se-
quenced to determine the positions of the intron/exon boundaries.
Smaller introns were sequenced in their entirety and exact sizes
determined. Sizes of larger introns (1–8 kb) were estimated by
comparison of PCR products to size standards. Introns 1 and 2
could not be amplified by PCR; therefore, intron/exon boundaries
were determined by direct sequencing of the PAC clones by using
Big-Dye technology (Applied Biosystems, UK). These intron sizes
were estimated by Southern blot hybridization of digested PAC
clones. Cacna2d2 is organized into 39 exons (Table 1 and Fig. 1A)
that are distributed over 85 kb of genomic DNA. Exon 1 contains
the start codon, and exon 39 contains the stop signal.
The mouse Cacna2d2 cDNA sequence was aligned with the
genomic sequence derived from a cosmid contig of human Chr
3p21.3 (GenBank Z84493, Z84494, Z84495, Z75743, Z75742, and
Z84492). The positions of the mouse intron/exon boundaries were
compared with regions of divergence between the mouse cDNA
sequence and the human genomic DNA sequence. The positions of
all the intron/exon boundaries and exon sizes are conserved be-
tween human and mouse, so CACNA2D2 is also organized into 39
exons over a genomic distance of at least 118 kb (Table 1 and Fig.
1B). Overall, the genomic organizations of the two genes are
Two regions of alternative splicing were identified in mouse
brain RNA (Fig. 2). Exon 23 (Fig. 2A) is present in 13% of
subclones of an RTPCR product spanning exons 22–24. At the
protein level, it results in the sequence change KYF to
KLPISKLKDF. A splice variant at the same position has been
described in human cDNA (Hobom et al. 2000). The 5Ј splice
donor does not conform to the consensus gt, but contains a gc in
both the mouse and human sequences (Table 1). Such a variation
at the 5Ј splice site has been previously described (Shapiro and
Senapathy 1987) and is not expected to significantly impair splic-
ing. Presumably, this represents a mechanism whereby the recog-
nition of the splice site is slightly reduced, consistent with the
relatively low abundance of RNA species containing exon 23.
The second region of alternative splicing involves the 3Ј splice
acceptor of intron 38. The 6 bp indicated (Fig. 2B) was identified
in 25% of subclones from an RTPCR product spanning exons
37–39 and results in the insertion of two amino acids (CP). Thus,
two 3Ј splice donor sites are associated with intron 37. The more
favored more 3Ј splice site contains the consensus ag motif. The
second splice site contains a non-consensus at motif, again sug-
gesting that divergence from the consensus produces alternatively
spliced products of relatively lower abundance. This non-
consensus splice site is conserved in humans, and the same splice
variant has been described in human medulla thyroid carcinoma
cells (Hobom et al. 2000).
Sequence analysis of full-length Cacna2d2 cDNA clones (n ס
9) and in silico analysis of cDNA sequences have not identified
transcripts that contain exon 23 and the 6-bp variant of exon 38.
Tissue-specific splicing of human CACNA2D2 has been de-
scribed, although functional expression of splice variants did not
reveal differences at the electrophysiological level (Hobom et al.
2000). Analysis at the protein level is required to provide definitive
proof that the alternatively spliced transcripts represent native pro-
Correspondence to: J. Barclay; E-mail: email@example.com
* Present address: Novartis Institute for Medical Sciences, 5 Gower Place,
London, WC1E 6BN, UK.
Mammalian Genome 11, 1142–1144 (2000).
© Springer-Verlag New York Inc. 2000