Novel Cav2.1 splice variants isolated from Purkinje cells do not generate P-type Ca2+ current.
The alpha(1)2.1 (alpha(1A)) subunits of P-type and Q-type Ca(2+) channels are encoded by a single gene, Cacna1a. Although these channels differ in the inactivation kinetics and sensitivity to omega-agatoxin IVA, the mechanism underlying these differences remains to be clarified. Alternative splicings of the Cacna1a transcript have been postulated to contribute to the respective properties, however, the splice variants responsible for P-type Ca(2+) channels have not been identified. To explore P-type-specific splice variants, we aimed at cloning alpha(1)2.1 from isolated mouse Purkinje cells using single-cell reverse transcription-PCR, because in Purkinje cells P-type currents dominate over the whole currents (>95%) with Q-type currents undetected. As a result, two novel splice variants were cloned. Compared with the previously cloned mouse alpha(1)2.1, two novel variants had additional 48 amino acids at the amino termini, six single amino acid changes, and splicing variations at the exon 46/47 boundary, which produced different carboxyl termini. Furthermore, one variant had one RNA editing site. However, electrophysiological and pharmacological studies indicated that these variants did not generate P-type current in cultured cells. These results suggest that P-type-specific splice variants may exist but that post-translational processing or modification by uncharacterized interacting proteins is also required for generating the P-type current.
Tsunemi
Taiji
T
Department of Pharmacology and Neurobiology, Graduate School of Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan.
Saegusa
Hironao
H
Ishikawa
Kinya
K
Nagayama
Shin
S
Murakoshi
Takayuki
T
Mizusawa
Hidehiro
H
Tanabe
Tsutomu
T
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D020864:Calcium Channels, N-Type / Q000235:genetics* / Q000378:metabolism*; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004594:Electrophysiology; D005091:Exons; D006801:Humans; D007700:Kinetics; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011689:Purkinje Cells / Q000378:metabolism*; D012313:RNA / Q000378:metabolism; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D000596:Amino Acids; D002118:Calcium; D020864:Calcium Channels, N-Type; D018076:DNA, Complementary; D012313:RNA