Reduction of insulin secretion in the insulinoma cell line INS-1 by overexpression of a Ca(v)2.3 (alpha1E) calcium channel antisense cassette.
OBJECTIVE
Multiple types of voltage-activated Ca(2+) channels (T, L, N, P, Q and R type) coordinate a variety of Ca(2+)-dependent processes in neurons and neuroendocrine cells. In insulinoma cell lines as well as in endocrine tissues, the non-L-type alpha1E (Ca(v)2.3) subunit is expressed as the tissue-specific splice variant alpha1Ee.


METHODS
To understand the functional role of alpha1E-containing Ca(2+) channels, antisense alpha1E mRNA was overexpressed in INS-1 cells by stable transfection of an antisense alpha1E cassette cDNA. As controls, either a sense alpha1E cassette or a control vector containing enhanced green fluorescent protein as an unrelated gene was stably transfected. The overexpression of each transfected cassette cDNA was recorded by RT-PCR.


RESULTS
In three independent antisense alpha1E INS-1 clones, the glucose-induced insulin release was significantly reduced as compared with wild-type INS-1 cells and with a sense alpha1E INS-1 clone. However, in the antisense INS-1 clones, the KCl-induced insulin release was less impaired by overexpressing the antisense alpha1E cassette than the glucose-induced insulin release, leading to the assumption that glucose (15 mmol/l) and KCl (25 mmol/l) finally depolarize the membrane potential to a different extent.


CONCLUSIONS
alpha1E is involved in glucose-induced insulin secretion probably by influencing the excitability of INS-1 cells.
Pereverzev
Alexey
A
Institute of Neurophysiology, University of Cologne, Robert Koch Strasse 39, D-50931 Cologne, Germany.
Vajna
Rolf
R
Pfitzer
Gabriele
G
Hescheler
Jürgen
J
Klöckner
Udo
U
Schneider
Toni
T
D015153:Blotting, Western; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D020908:Calcium Channels, R-Type; D027682:Cation Transport Proteins; D016373:DNA, Antisense / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D049452:Green Fluorescent Proteins; D006801:Humans; D007202:Indicators and Reagents; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007340:Insulinoma / Q000378:metabolism*; D008164:Luminescent Proteins; D016254:Mutagenesis, Insertional; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014407:Tumor Cells, Cultured; D015854:Up-Regulation
C423009:CACNA1E protein, human; D015220:Calcium Channels; D020908:Calcium Channels, R-Type; D027682:Cation Transport Proteins; D016373:DNA, Antisense; D018076:DNA, Complementary; D007202:Indicators and Reagents; D007328:Insulin; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins