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Appearance of voltage-gated calcium channels following overexpression of ATPase II cDNA in neuronal HN2 cells.
- Source :
-
Brain research. Molecular brain research [Brain Res Mol Brain Res] 2003 Oct 07; Vol. 117 (2), pp. 109-15. - Publication Year :
- 2003
-
Abstract
- ATPase II (a Mg2+-ATPase) is also believed to harbor aminophospholipid translocase (APTL) activity, which is responsible for the translocation of phosphatidylserine (PS) from the outer leaflet of the plasma membrane to the inner. To test this hypothesis we overexpressed the mouse ATPase II cDNA in the neuronal HN2 cells. In addition to a dramatic increase in APTL activity, we also made the unexpected observation that expression of the mouse ATPase II cDNA from the vector pCMV6 resulted in the appearance of calcium current. Although the hybrid cell line HN2 or a line (HN2V32) obtained by expressing a heterologous gene from the same expression vector showed no calcium current, both ATPase II-overexpressing clones (HN2A12 and HN2A22) showed significant barium conductance. This current was due to calcium channels because it was blocked almost completely by 100 microM CdCl2 and it had a significant N-type component since it was blocked by 38.5% in the presence of 5 microM omega-conotoxin (omega-CTX). Western blot analysis using an antibody against the N-type calcium-channel alpha1B subunit revealed a dramatic increase in expression of this protein in the HN2A12 and HN2A22 cell lines. Our results suggest that ATPase II also harbors APTL activity. In view of the prior knowledge that APTL activity is inhibited by an increase in calcium, our results also suggest that APTL expression exerts a negative feedback regulation on itself by inducing expression of channels that cause an influx of calcium ions. The mechanism of this regulation could reveal important information on a possible cross-regulation between these two families of proteins in neuronal cells.
- Subjects :
- Adenosine Triphosphatases genetics
Blotting, Western
Cadmium Chloride pharmacology
Calcium Channel Blockers pharmacology
Calcium Channels, N-Type genetics
Carrier Proteins metabolism
Cell Line
Hippocampus cytology
Hippocampus metabolism
Membrane Potentials drug effects
Membrane Proteins metabolism
Mitogen-Activated Protein Kinase 1 metabolism
Mitogen-Activated Protein Kinase 3
Mitogen-Activated Protein Kinases metabolism
Neuroblastoma
Neurons drug effects
Patch-Clamp Techniques
Protein Isoforms metabolism
Transfection
omega-Conotoxins pharmacology
Adenosine Triphosphatases metabolism
Calcium Channels, N-Type metabolism
DNA, Complementary metabolism
Neurons metabolism
Phospholipid Transfer Proteins
Subjects
Details
- Language :
- English
- ISSN :
- 0169-328X
- Volume :
- 117
- Issue :
- 2
- Database :
- MEDLINE
- Journal :
- Brain research. Molecular brain research
- Publication Type :
- Academic Journal
- Accession number :
- 14559144
- Full Text :
- https://doi.org/10.1016/s0169-328x(03)00210-9