Novel variants of voltage-operated calcium channel alpha 1-subunit transcripts in a rat liver-derived cell line: deletion in the IVS4 voltage sensing region.
Using reverse transcriptase-PCR and Northern analysis, we have shown that the H4IIE cell line, derived from the Reuber H35 rat hepatoma, contains significant amounts of transcripts for the CaCh3 (neuroendocrine) and CaCh1 (skeletal muscle) L-type voltage-operated calcium channel alpha 1-subunits. Two of the CaCh3 transcripts have a 45 bp deletion in the IVS4 membrane-spanning region which is the result of a mutation in genomic DNA. The deduced amino acid sequences of the PCR-derived clones of CaCh3 indicate that the mutation causes the loss of 15 amino acids from the IVS4 region, including three of the six positively charged residues, which are thought to be part of the voltage-sensing mechanism of voltage-operated Ca2+ channels. Quantitative-PCR and Northern analysis indicate that one of the novel CaCh3 transcripts is present in sufficient amounts to imply it could play a functional role in Ca2+ inflow. RT-PCR analysis of hepatocytes isolated from rat liver detected transcripts of CaCh3 (without the IVS4 mutation) and CaCh2, but at considerably lower levels than observed for the isoforms in the H4IIE cell line. Transcripts of CaCh1 and CaCh2 were also detected at low levels in Jurkat T lymphocytes. Fluorimetric studies with the Ca(2+)-sensitive probe, Fluo-3, have shown that H4IIE cells exhibit receptor-activated and store-activated (thapsigarin-induced), but not depolarisation (extracellular KCl)-induced Ca2+ inflow. The mutant transcripts are unlikely to produce Ca2+ channels that are opened by membrane depolarisation. The idea that they may be opened by other mechanisms is briefly discussed.
Brereton
H M
HM
Department of Medical Biochemistry, School of Medicine, Flinders University, Adelaide, Australia.
Harland
M L
ML
Froscio
M
M
Petronijevic
T
T
Barritt
G J
GJ
D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D015220:Calcium Channels / Q000235:genetics* / Q000378:metabolism*; D020746:Calcium Channels, L-Type; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D017931:DNA Primers; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008099:Liver / Q000737:chemistry / Q000166:cytology* / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction; D012326:RNA Splicing; D051381:Rats; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D019284:Thapsigargin / Q000494:pharmacology; D014158:Transcription, Genetic
D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D017931:DNA Primers; D004791:Enzyme Inhibitors; D019284:Thapsigargin