Appearance of voltage-gated calcium channels following overexpression of ATPase II cDNA in neuronal HN2 cells.
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.
Chin
Gary
G
Department of Chemistry and the CSI/IBR Center for Developmental Neuroscience, City University of New York at the College of Staten Island, Staten Island, New York, NY 10314, USA.
El-Sherif
Yasir
Y
Jayman
Farah
F
Estephan
Rima
R
Wieraszko
Andrzej
A
Banerjee
Probal
P
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D019256:Cadmium Chloride / Q000494:pharmacology; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020864:Calcium Channels, N-Type / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D018076:DNA, Complementary / Q000378:metabolism*; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects; D008565:Membrane Proteins / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009447:Neuroblastoma; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D049294:Phospholipid Transfer Proteins; D020033:Protein Isoforms / Q000378:metabolism; D014162:Transfection; D020960:omega-Conotoxins / Q000494:pharmacology
D002121:Calcium Channel Blockers; D020864:Calcium Channels, N-Type; D002352:Carrier Proteins; D018076:DNA, Complementary; D008565:Membrane Proteins; D049294:Phospholipid Transfer Proteins; D020033:Protein Isoforms; D020960:omega-Conotoxins; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D000251:Adenosine Triphosphatases; D019256:Cadmium Chloride