Isoforms of α1E voltage-gated calcium channels in rat cerebellar granule cells -: Detection of major calcium channel α1-transcripts by reverse transcription-polymerase chain reaction

In primary cultures of rat cerebellar granule cells, transcripts of voltage-gated Ca2+ 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 alpha 1C, alpha 1A, and alpha 1E are detected using degenerated oligonucleotide primer pairs under highly stringent conditions. Whole-cell Ca2+ 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%) Ca2+; channels. These data support the correlation between alpha 1A and P-type Ca2+ channels (G1) and between alpha 1E and R-type channels (G2 and G3). By including specific primer pairs for alpha 1E the complimentary DNA fragments of indicative regions of alpha 1E isoforms are amplified corresponding to the three most variable regions of alpha 1E, 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 alpha 1E 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 alpha 1E complementary DNA. It corresponds to the alpha 1E 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 alpha 1E. While fragments of a new rat alpha 1E 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 alpha 1E expression from a pattern seen in cerebellum (alpha 1Ee) to a pattern identified in other regions of the brain (alpha 1E-3) is discussed. These data show that: (i) alpha 1E is expressed in rat brain as a structural homologue to the mouse and human alpha 1E; and (ii) rat cerebellar granule cells in primary culture express a set of alpha 1E isoforms, containing two different sized carboxy termini. Since no new transcripts of high-voltage-activated Ca2+ 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. (C) 1999 IBRO. Published by Elsevier Science Ltd.