Expression of a cDNA for a neuronal calcium channel alpha 1 subunit enhances secretion from adrenal chromaffin cells.
A synthetic oligonucleotide was used to isolate mouse brain cDNA clones coding for a brain isoform of the alpha 1 subunit of the voltage-sensitive Ca2+ channel. Twenty-six independent cDNA clones were isolated and sequenced. All the cDNA clones reported here showed high homology to the rat brain class C cDNA sequence (Snutch, T. P., Tomlinson, W. J., Leonard, J. P., and Gilbert, M. M. (1991) Neuron 7, 45-57). Comparison of the individual mouse brain class C (mbC) cDNA sequences indicated the presence of four regions within the alpha 1 subunit coding sequence where alternative splicing can take place in mouse brain and raise the possibility that combinatorial arrangement of these splice variants could give rise to a heterogenous class of mbC transcripts. Northern blot analysis demonstrated that mbC mRNA sequences could be detected in highest abundance in mouse heart, at lower levels in mouse brain and spinal cord, and not at all in liver or skeletal muscle. An expression vector for one isoform of the mbC alpha 1 subunit cDNA was constructed using the human cytomegalovirus promoter to direct expression, and this expression vector was used in a novel transfection assay of primary cultures of bovine adrenal chromaffin cells. Transfection of the mbC alpha 1 subunit expression vector increased the secretion of human growth hormone derived from a cotransfected human growth hormone expression vector after stimulation with elevated K+ or the dihydropyridine agonist, Bay K8644. These experiments suggest that this isoform of the mbC alpha 1 subunit is functional in the transfected chromaffin cells and that the number of Ca2+ channels is a limiting component in the secretion from chromaffin cells in culture.
Ma
W J
WJ
Shanghai Institute of Materia Medica, Chinese Academy of Science.
Holz
R W
RW
Uhler
M D
MD
D000313:Adrenal Medulla / Q000187:drug effects / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015220:Calcium Channels / Q000235:genetics* / Q000502:physiology*; D002395:Catecholamines / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D005711:Gallopamil / Q000494:pharmacology; D015870:Gene Expression; D005822:Genetic Vectors; D046911:Macromolecular Substances; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D009132:Muscles / Q000502:physiology; D009838:Oligodeoxyribonucleotides; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D014162:Transfection
D015220:Calcium Channels; D002395:Catecholamines; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D011994:Recombinant Proteins; D005711:Gallopamil; D012313:RNA; D004247:DNA