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In primary cultures of rat cerebellar granule cells, transcripts of voltage-gated Ca 2+ channels have been amplified by reverse transcription–polymerase chain reaction and identified by sequencing of subcloned polymerase chain reaction products. In these neurons cultured for six to eight days in vitro , fragments of the three major transcripts α1C, α1A, and α1E are detected using degenerated oligonucleotide primer pairs under highly stringent conditions. Whole-cell Ca 2+ current recordings from six to eight days in vitro granule cells show that most of the current is due to L-type (25%), P-type (33%) and R-type (30%) Ca 2+ channels. These data support the correlation between α1A and P-type Ca 2+ channels (G1) and between α1E and R-type channels (G2 and G3). By including specific primer pairs for α1E the complimentary DNA fragments of indicative regions of α1E isoforms are amplified corresponding to the three most variable regions of α1E, the 5′-end, the II/III-loop, and the central part of the 3′-end. Although the complementary DNA fragments of the 5′-end of rat α1E yield a uniform reverse transcription–polymerase chain reaction product, its structure is unusual in the sense that it is longer than in the cloned rat α1E complementary DNA. It corresponds to the α1E isoform reported for mouse and human brain and is also expressed in cerebellum and cerebrum of rat brain as the major or maybe even the only variant of α1E. While fragments of a new rat α1E isoform are amplified from the 5′-end, three known fragments of the II/III-loop and two known isoforms homologue to the 3′-coding region are detected, which in the last case are discriminated by a 129 base pair insertion. The shift of the α1E expression from a pattern seen in cerebellum (α1Ee) to a pattern identified in other regions of the brain (α1E-3) is discussed. These data show that: (i) α1E is expressed in rat brain as a structural homologue to the mouse and human α1E; and (ii) rat cerebellar granule cells in primary culture express a set of α1E isoforms, containing two different sized carboxy termini. Since no new transcripts of high-voltage-activated Ca 2+ channels genes are identified using degenerate oligonucleotide primer pairs, the two isoforms differentiated by the 129 base pair insertion might correspond to the two R-type channels, G2 and G3, characterized in these neurons. Functional studies including recombinant cells with the different proposed isoforms should provide more evidence for this conclusion. |
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