Elsevier

Molecular Brain Research

Volume 117, Issue 2, 7 October 2003, Pages 109-115
Molecular Brain Research

Research report
Appearance of voltage-gated calcium channels following overexpression of ATPase II cDNA in neuronal HN2 cells

https://doi.org/10.1016/S0169-328X(03)00210-9Get rights and content

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 μM CdCl2 and it had a significant N-type component since it was blocked by 38.5% in the presence of 5 μM ω-conotoxin (ω-CTX). Western blot analysis using an antibody against the N-type calcium-channel α1B 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.

Introduction

In normal cells, the aminophosphospholipids, phosphatidylserine (PS) and phosphatidyl-ethanolamine (PE) are sequestered to the inner leaflet of the plasma membrane [2], [11], [14], [17], [28], [30], [31]. This is caused by an enzyme activity, termed aminophospholipid translocase (APTL), which uses energy derived from ATP hydrolysis to drive translocation of both PS and PE from the outer to the inner leaflet of the plasma membrane [31]. However, PS is translocated much faster, with half times of 5–10 min, using one ATP molecule per molecule of lipid translocated [4], [6], [20]. The lipid translocase activity is Mg2+-dependent, and is inhibited by vanadate, high intracellular calcium, and the sulfhydryl-modifying agent N-ethylmaleimide [29], [31]. In addition to APTL, there are two more known enzyme activities that regulate transmembrane lipid movement. The first one is a less specific ATP-dependent floppase activity that translocates both aminophospholipids as well as the choline phospholipids from the inside to the outside at a rate 10 times slower [6]. The second enzyme is a Ca2+-dependent enzyme, scramblase, which causes non-specific flip-flop of phospholipids from one layer to the other (the inner or the outer leaflet) of the plasma membrane [31]. Thus, a combined and balanced action of the enzymes, translocase, floppase, and scramblase, seems to equip the cell with the ability to correct for alterations in lipid distributions to avoid potential consequences of having essential cells being phagocytosed by scavenger cells [7], [31].

The mammalian enzyme ATPase II that belongs to a family of P-type ATPases has strikingly similar properties to those described above for the aminophospholipid translocase [14], [28]. Furthermore, a mutant yeast strain lacking the expression of a gene (Drs2) that is homologous to mammalian ATPase II showed inhibited translocation of PS across the plasma membrane [28]. Based on such reports it is currently believed that ATPase II is probably an APTL [14], [28]. Mammalian P-type ATPases occur at high levels throughout the central nervous system (CNS) [13], [14]. The ATPase II protein was initially cloned from secretory vesicles of the bovine adrenal medulla chromaffin granules, where this enzyme is expressed at a high level [28]. In later studies, the mouse and human homologues of this gene were also cloned and their sequences published [14], [21].

During apoptosis, the lipid asymmetry of the plasma membrane is lost, probably due to an inhibition of APTL. Recent studies have shown that inhibition of the aminophospholipid translocase activity is essential for the characteristic apoptosis-associated externalization of PS [5], [12]. Also we have observed that APTL activity is inhibited during apoptosis in the HN2-derived HN2-5 and human oligodendroglioma (HOG) cells [8]. Our studies have also shown that PS is externalized in the apoptotic HN2-5 cells, which then triggers phagocytosis of these cells by ameboid microglia [2]. The mammalian body is continuously replacing old cells for new. The old and unwanted cells undergo apoptosis and are rapidly phagocytosed by scavenger cells such as macrophages. The externalized PS molecules on apoptotic cells are recognized by the phagocytic cells, which harbor a set of cell surface proteins/molecules that bind with high affinity to PS [2], [11], [14], [28], [30], [31]. One such PS receptor has been cloned recently and it is expected that other similar proteins exist in nature [10].

Inhibition of APTL activity by an increase in either extracellular or intracellular Ca2+ concentration [5], [30] suggests that the plasma membrane calcium channels, which mediate an influx of Ca2+ ions into a cell, could regulate the APTL activity. Based on electrophysiological and pharmacological properties, these channels have been classified into five major groups, L, N, T, R, and P/Q types [18], [19]. In a typical patch-clamp analysis, if the membrane potential is first clamped at −100 mV and then stepped up in a pulse to +20 mV, then all the Ca2+ channel types are opened at once, thus confirming their voltage-gated nature. Subsequent passive entry of Ca2+ ions from a higher extracellular concentration into cytosol, results in a variety of physiologic effects depending on tissue-specific expression of the individual channel types.

Our initial idea was to test the hypothesis ‘ATPase II is an APTL’ by overexpressing the murine ATPase II cDNA in the mouse hippocampal neuron-derived cell line, HN2. However, an unexpected observation was made during the course of this project. The HN2 cells that originally displayed no calcium current, yielded significant levels of voltage-gated calcium current following transfection. A significant fraction of this mixed current was due to the N-type channels. While the details of the effect of overexpression of ATPase II on PS translocation in HN2 cells will be included in a different publication, the purpose of this article is to report our surprising observation linking one putative plasma membrane protein (ATPase II) to the expression and activity of another family of membrane proteins, the voltage-gated calcium channels. This suggests that the protein ATPase II may play an important role in regulating the expression and/or activity of calcium channels.

Section snippets

Preparation of APTL-overexpressing cell lines

The murine ATPase II cDNA sequence (GeneBank accession number: U75321) in the original vector (pBluescript) (a kind gift from Robert Schlegel) was cleaved by digestion with XbaI and SalI and inserted between XbaI and SalI sites on the mammalian expression vector pCMV6c. The recombinant construct obtained (pCMV-ATPase II) was linearized by digestion at a PvuI site within the β-lactamase gene present on the same vector and then transfected into the hippocampal neuron-derived, hybrid neuroblastoma

Overexpression of ATPase II in the neuronal cell line HN2

We transfected the mouse ATPase II cDNA into the mouse HN2 cells and subjected the transfected cells to Geneticin selection to prepare clones overexpressing ATPase II with the initial intention to study its effect on APTL activity. Although our assays indicated that a large number of the selected clones had increased APTL activity, two clones, HN2A12 and HN2A22, presented here, showed the maximum increase, i.e. a fivefold elevation in APTL activity as compared to that in the untransfected HN2

Discussion

Gleiss and co-workers have recently demonstrated that between Jurkat and Raji cells the latter cell line expresses higher levels of ATPase II mRNA [12]. Upon treatment of both cell lines with the thiol-modifying agent N-ethylmaleimide (known to inactivate APTL), the Jurkat cells, which showed lower levels of ATPase II mRNA, also displayed more rapid externalization of PS. Moreover, upon treatment with anti-Fas antibody, which triggers apoptosis in these cells, APTL activity (measured by

Acknowledgements

We wish to express our gratitude to Drs Robert Schlegel (Pennsylvania State University), Vanda Lenon (Mayo Clinic, Rochester, MN), and Xiao-Song Xie (UT Southwestern Medical Center) for their kind gifts of mouse APTL cDNA, α1B antibody, and the ATPase II antibody, respectively. Yasir El-Sherif was supported in part by an OMRDD fellowship in the CSI/IBR Center for Developmental Neuroscience and Developmental Disabilities. Farah Jayman and Rima Estephan were supported in part by fellowships from

References (31)

Cited by (6)

  • Isolation, sequencing, and functional analysis of the TATA-less murine ATPase II promoter and structural analysis of the ATPase II gene

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    Our previous studies have shown that apoptosis is associated with an inhibition of APLT and externalization of PS [7,10]. We have also demonstrated that overexpression of ATPase II in the hybrid neuroblastoma cell line HN2 causes an increase in APTL activity [17], which suggests the possibility that ATPase II is indeed involved in PS translocation. During apoptosis, many other genes and their products are regulated to cause signature changes that are observed in the apoptotic cells.

  • Isolation, sequencing, and functional analysis of the TATA-less human ATPase II promoter

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1

Gary Chin and Yasir El-Sherif contributed equally toward the project.

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