Identification of the alternative spliced form of the alpha 2/ delta subunit of voltage sensitive Ca super(2+) channels expressed in PC12 cells

The alpha 2/ delta subunit of voltage sensitive Ca super(2+) channels expressed in PC12 has been cloned and partially sequenced. The message observed in Northern blot analysis displays a 7.5 kb transcript, identical in size to mRNA of rabbit skeletal muscle and rat brain. The nucleotide sequence of the cloned alpha 2 subunit of the PC12 specific cDNA is >99% identical to rat brain sequence and 85% to skeletal muscle. Reverse-transcriptase-polymerase chain reaction (RT-PCR) of the alternative splicing region identifies two deleted regions of 57 bp and 21 bp in PC12 expressed alpha 2/ delta transcript. The alternative variant alpha sub(2e) of alpha 2/ delta subunit which is expressed in PC12 cells was previously identified in human embryonic kidney (HEK293) cells. RT-PCR analysis show two different sized alternative PCR fragments in rat lung and none in rat spleen, kidney and intestine. Antibodies prepared against a 19 amino acid peptide within the alternative spliced region effectively inhibits [ super(3)H]dopamine release in PC12 cells. This implies that the alternatively spliced region is positioned extracellularly and is involved in regulation of the L-type Ca super(2+) channel-mediated transmitter release.