Original Article
Identification and Cloning of Putative Human Neuronal Voltage-Gated Calcium Channel γ-2 and γ-3 Subunits: Neurologic Implications

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Objective

To identify human genes encoding neuronal voltage-gated calcium channel γ subunits corresponding to the γ subunit recently described in mice with hereditary epilepsy, confirm their expression in adult brain, and determine whether a γ subunit resides in human chromosomal regions with marker genes linked to convulsions.

Material and Methods

Human homologues of a recently described mouse γ subunit were sought by a computerized search of National Center for Biotechnology Information databases. Sequences of interest were mapped electronically to specific chromosomal regions. The proximity of these chromosomal regions to markers linked to convulsive disorders was determined by a MEDLINE search. Predicted γ subunit sequences were amplified by polymerase chain reaction from a human adult brain complementary DNA (cDNA) library and were subcloned, and full-length sequences were determined or confirmed. Secondary structure and transmembrane regions were predicted by using the TMpredict program.

Results

Two putative isoforms (human γ-2 and γ-3 calcium channel subunits) were identified, cloned, and sequenced. They mapped to chromosomes 22 and 16, respectively. A marker very close to the γ-3 gene gives maximal lod scores for an autosomal dominant syndrome of familial infantile convulsions and paroxysmal choreoathetosis.

Conclusion

Genomic DNA sequence data already existed in GenBank (partial for γ-2 and complete for γ-3) for the two putative neuronal calcium channel γ subunits that we identified. By cloning both of these molecules from a cerebellar cDNA library, we demonstrated for the first time their expression in human brain. The γ-3 gene is adjacent to a marker for a convulsive disorder. The convulsive phenotype of γ channelopathies occurring in mice makes the human γ subunit genes attractive candidate genes for hereditary convulsive disorders.

Section snippets

Computerized Analysis of GenBank and MEDLINE Databases

Because several channelopathies have been identified in hereditary seizure disorders of humans and mice,3, 4, 5, 6, 7, 8, 9, 10 we used the National Center for Biotechnology Information (NCBI) Gapped BLAST11 search program to look for human homologues for the recently described mouse γ-2 sequence.1 We used the NCBI programs Electronic PCR12 and Map Search13, 14 to localize amino acid sequences of interest to specific chromosomal regions, and a software program called TMpredict15 was used to

RESULTS

Using a cDNA library derived from adult human cerebellum RNA, we cloned and fully sequenced the human γ-2 cDNA noted by Letts and associates1 as a partial sequence on chromosome 22 (GenBank accession numbers Z83733 and AL022313). We then used the amino acid sequence that Letts and colleagues1 established for the mouse γ-2 subunit to identify, by computer-assisted searching of NCBI resources, a previously unrecognized homologous sequence from Homo sapiens on chromosome 16p12p13.1, which is

DISCUSSION

On the basis of this study, we conclude that heteromeric voltage-gated calcium channels in human neurons likely contain γ subunits in addition to their previously recognized a.I, α2-8, and ˜ subunits. Before publication of the report by Letts and associates,1 a γ subunit (γ-1) was recognized only in the dihydropyridine-sensitive L-type calcium channel found in skeletal muscle. Two genomic sequences that had been deposited in GenBank databases, and are the subject of this report (&gammal-2 noted

CONCLUSION

Further developmental, mutational, and immunologic studies of the human γ-2 and γ-3 neuronal calcium channel subunit genes are warranted. In particular, these genes merit surveillance for mutations in persons with familial infantile convulsions and paroxysmal choreoathetosis. The dominant γ subunit isoform expressed at various stages of development and in all brain regions should be determined as well because these genes may be preferentially expressed at different stages of development, as is

ACKNOWLEDGMENT

We thank Dr. Daniel J. McCormick, director of the Mayo Clinic Protein Core Facility, for performing amino acid sequence comparison and consensus analyses.

REFERENCES (17)

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This study was supported in part by funds from the Mayo Foundation (J.L.B.) and Grant CA 37343 from the National Cancer Institute (V.A.L).

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