Novel Cav2.1 Splice Variants Isolated from Purkinje Cells Do Not Generate P-type Ca2+ Current

Abstract

The ␣ 1 2.1 (␣ 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 -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-typespecific splice variants, we aimed at cloning ␣ 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 ␣ 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.

Spinocerebellar ataxia type 6 knockin mice develop a progressive neuronal dysfunction with age-dependent accumulation of mutant Ca V 2.1 channels

“…S1C]. At the boundary of mouse intron 46/exon 47, we confirmed the presence of the sequence AGGGCAGTAG, in which each of the three AG sequences can serve as a splice acceptor site to generate distinct isoforms (12) (Fig. S1 A and B).…”

“…Clones obtained from the 129/SvEv mouse genomic DNA library were used to construct a targeting vector. A SacII fragment that includes part of the mouse Sca6 exon 47 was replaced with the homologous fragment prepared from human Ca V2.1 cDNAs (12). A selectable cassette containing the neomycin resistance gene (Neo) and the thymidine kinase gene (Tk) flanked by two loxP sites were inserted into intron 45.…”

Systematic Identification of Splice Variants in Human P/Q-Type Channel α12.1 Subunits: Implications for Current Density and Ca2+-Dependent Inactivation

“…Splice variation of ␣ 1A (␣ 1 2.1) channels could have important functional correlates, as already shown by previous studies of partial sets of variants (Bourinet et al, 1999;Hans et al, 1999;Krovetz et al, 2000;Tsunemi et al, 2002). The more exhaustive suite of variants delineated here by systematic exon scanning raises expectations that splice-related functional diversity may well be considerably larger than currently appreciated.…”

“…1C) was designed against the 3Ј untranslated region of the known exon 47. These reactions would detect use of alternate splice acceptordonor sites with the known exons (e.g., ⌬47/47) but not of potential alternate versions of exons 1 and 47 (Tsunemi et al, 2002). Also, rare cell-specific RNA editing restricted to certain cells (Tsunemi et al, 2002) could elude our screen of cell population libraries.…”

Development of Purkinje cell degeneration in a knockin mouse model reveals lysosomal involvement in the pathogenesis of SCA6

“…In SCA6, the CAG repeat encodes a polyQ tract in the cytoplasmic tail domain of the Ca v 2.1 channel, a pore-forming subunit of the P/Q type voltage-gated calcium channels (6). Alternative splicing occurring at the beginning of the final exon (exon 47) of CAC-NA1A produces two major Ca v 2.1 isoforms, MPI and MPc (7). These two isoforms differ in their splice acceptor sites, such that MPI translates a polyQ tract from the CAG coding repeat, whereas MPc splices to an immediate stop codon, resulting in a shorter cytoplasmic tail that lacks the polyQ tract.…”