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

Molecular Brain Research 117 (2003) 109–115 www.elsevier.com / locate / molbrainres

Research report

Appearance of voltage-gated calcium channels following overexpression of ATPase II cDNA in neuronal HN2 cells Gary Chin 1 , Yasir El-Sherif, Farah Jayman, Rima Estephan, Andrzej Wieraszko, Probal Banerjee* 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

Abstract ATPase II (a Mg 21 -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 mM CdCl 2 and it had a significant N-type component since it was blocked by 38.5% in the presence of 5 mM v-conotoxin (v-CTX). Western blot analysis using an antibody against the N-type calcium-channel a 1B 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.  2003 Elsevier B.V. All rights reserved. Theme: Excitable membranes and synaptic transmission Topic: Calcium channel structure, function, and expression Keywords: Flippase; Calcium channels; Aminophospholipid translocase; Phosphatidylserine; ATPase II

1. 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

Abbreviations: APTL, aminophospholipid translocase; v-CTX, vconotoxin; HBS, HEPES buffered saline *Corresponding author. Tel.: 11-718-982-3938; fax: 11-718-9823944. E-mail address: [email protected] (P. Banerjee). 1 Gary Chin and Yasir El-Sherif contributed equally toward the project. 0169-328X / 03 / $ – see front matter  2003 Elsevier B.V. All rights reserved. doi:10.1016 / S0169-328X(03)00210-9

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 Mg 21 -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 Ca 21 -dependent enzyme, scramblase, which causes nonspecific 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

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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 Ca 21 concentration [5,30] suggests that the plasma membrane calcium channels, which mediate an influx of Ca 21 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 2100 mV and then stepped up in a pulse to 120 mV, then all the Ca 21 channel types are opened at once, thus confirming their voltage-gated nature. Subsequent passive entry of Ca 21 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.

2. Materials and methods

2.1. 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 (pCMVATPase II) was linearized by digestion at a PvuI site within the b-lactamase gene present on the same vector and then transfected into the hippocampal neuron-derived, hybrid neuroblastoma cell line HN2 by electroporation at 200 V and 975 mF. The transfectants were selected in 400 mg / ml G418 and the clones obtained were screened by APTL assays. Two clones expressing high levels of APTL activity (HN2A12 and HN2A22) were discussed in this report.

2.2. Aminophospholipid translocase assay APTL assays were conducted according to reported procedures [15,29]. Briefly, the cells were washed twice with HEPES buffered saline (HBS) (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.32 mM Na 2 HPO 4 , 1.3 mM CaCl 2 , 0.8 mM MgSO 4 , 5.5 mM glucose) and then incubated at 37 8C in 2 ml of HBS for 5 min. Following this, 1-ml vesicles containing 20 mM NBD-PS (h1-oleoyl2-C6-[7-nitro-2-oxa-1,3-diazol-4-ylj amino] caproy1-snglycero-3-phosphoserine) (Avanti Polar Lipids, Inc.) and 40 mM PC were added to the overlayer of 2 ml HBS. After gentle mixing, the cells were incubated at 37 8C for 10 min and then the plates were placed on ice. Next, 1 ml of an ice-cold solution of 2% bovine serum albumin (BSA) in Krebs Ringer buffer (KRB) (25 mM HEPES, 20 mM

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glucose, 5 mM KCl, 1.28 mM CaCl 2 , 1.2 mM MgCl 2 , 145 mM NaCl, pH 7.4) was added to each plate followed by aspiration. The cells were then incubated on ice for 5 min with an additional 2 ml of ice cold KRB plus 2% BSA. This process of back extraction with BSA solution was repeated two more times to wash off all the NBD-PS that was not flipped to the inner leaflet by APTL. The cells were next washed three times with ice-cold phosphate buffered saline (PBS) (3 ml / wash) and then harvested in ice-cold PBS. After cell counting, aliquots containing equal numbers of cells from each sample were added in a total volume of 100 ml to the wells of a 96-well plate for fluorescence reading. Each sample was read in triplicate using a fluorescence plate reader set at excitation and emission wavelengths of 485 and 530 nm, respectively.

2.3. SDS-PAGE and immunoblot analysis The cell or tissue pellets were homogenized on ice in RIPA buffer (PBS containing 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 0.5 mM Na 3 VO 4 plus freshly added protease inhibitor cocktail; Boeringer) and then assayed for protein concentration. For the analysis of calcium channel a 1B subunit, aliquots containing 150 mg protein from each sample were mixed with an equal volume of SDS-PAGE treatment buffer and incubated on ice for 15–20 min followed by resolution on 7–16% gradient SDS–polyacrylamide gel. The resolved proteins were transferred to nitrocellulose membrane and then probed with an a 1B monoclonal antibody [25]. The nitrocellulose membrane was blocked overnight (at 4 8C) with 10% milk in 0.1% Tween-PBS, then treated with 1:200 dilution of the a 1B antibody in fresh blocking solution, washed three times with Tween-PBS, treated with per-

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oxidase-linked anti-mouse IgG (1:1000), washed, and then the protein bands were detected using the Super Signal Dura kit (Pierce). Following this, the blots were stripped by incubating at room temperature for 1 h in a stripping buffer (0.25 M glycine, pH 2.0), blocked again and then probed with 1:1000 dilution of a polyclonal ERK1 / 2 antibody (Santa Cruz Biotech, CA). The secondary antibody concentration maintained in this case was 1:50,000. For the immunodetection of ATPase II the samples were placed in the treatment buffer and then heated in a boiling water bath for 5 min. After SDS-PAGE and transfer to nitrocellulose membrane, a rabbit polyclonal antibody directed toward the N-terminal region of ATPase II (antibody B; reference number 2616) was used at 1:10,000 dilution [9]. The same secondary antibody, as used for the immunodetection of ERK1 / 2, was applied also for the detection of ATPase II.

2.4. Electrophysiology The pipette solution used to measure Ca 21 current was composed of (in mM): CsCl (110), HEPES (40), EGTA (10), MgATP (4), MgCl 2 (1), GTP (0.3), phosphocreatine (14): pH adjusted to 7.3 with CsOH; 290–295 mOsm [1]. The extracellular solution was continually perfused at a rate of about 2 ml / min into a bath containing about 1 ml of recording solution. The external recording solution, designed to isolate calcium channel currents (carried by Ba 21 ), contains the following (in mM): TEACl (120), BaCl 2 (30), HEPES (10), sucrose (20): pH adjusted to 7.3 with TEAOH; 315–318 mOsm. The contribution of Na 1 ions was blocked by the addition of 0.1 mM tetrodotoxin (TTX) to all external solutions. Drugs that dissolve in the extracellular solution were added to the perfusate.

Fig. 1. Stable overexpression of ATPase II in HN2 cells. (a) Aminophospholipid translocase assays showed a greater than fivefold increase in activity in the stable transfectants HN2A12 and HN2A22. Results are shown after protein normalization and are the mean of four independent determinations with duplicate samples. (b) Western blot analysis using an N-terminal directed antibody also showed a dramatic increase in ATPase II expression (114 kDa) in HN2A12 (lane 2) and HN2A22 (lane 3) as compared to that in the HN2 cells (lane 1). Normalization was carried out with respect to ERK2 band intensities obtained by reprobing of the same blot using ERK1 / 2 antibody. Each lane received 100 mg of cell lysate protein (P,0.025 between HN2 and HN2A12 and also between HN2 and HN2A22).

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An Axopatch 200A patch clamp amplifier was used to voltage-clamp undifferentiated neurons with a cell soma of at least 20 mm diameter; using the whole cell configuration. Electrodes, pulled from soda-lime glass capillary tubes ranged in resistance from 1.8 to 2.5 MV. The series resistance circuit of the amplifier was used to compensate 75% of the apparent series resistance. Clamp settling time was typically less than 300 ms. When measuring Ca 21 currents in TEA, the seal resistance often diminished by 2–4 GV. For all current traces the voltage was held at 2100 mV for 10 ms and then increased to 120 mV for 200 ms and back down to 2100 mV for 10 ms. The duration between each episode was 5 s, which was sufficient to prevent current run down. During the experiment, at regular periods, we obtained leak sweeps. Leak sweeps consisted of 16 averaged hyperpolarizing steps of 10 mV. The leak sweep currents were subtracted from the individual current recordings, offline. The data were filtered at 2 KHz then digitized at |100 ms per data point. Voltage protocols were generated and analyzed by an IBM PC Pentium clone using the PClamp6 software and the resultant data were analyzed and prepared using both PClamp6 and SigmaPlot 4.0.

3. Results

3.1. 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 cells (Fig. 1a). A significant increase in ATPase II expression was recorded in the HN2A12 and HN2A22 cells also by Western blot analysis using an N-terminaldirected antibody (Fig. 1b) [9]. Reprobing with an ERK1 / 2 antibody, densitometric normalization of ATPase II band intensities to ERK2 and then averaging of data over three determinations (n53) showed a significant increase in the 114-kDa ATPase II band in both HN2A12 and HN2A22 cells (Fig. 1b, lowest panel).

3.2. ATPase II-overexpressing clones displayed significant levels of voltage-gated calcium current, whereas the parent HN2 cells did not Patch-clamp electrophysiology showed that although the

Fig. 2. Appearance of voltage-gated Ca 21 current in clones overexpressing APTL. (A) Parent HN2 cells show no Ca 21 current. Although data shown here were collected with HNA12 and HN2A22, other ATPase II-overexpressing clones were also found to express similar voltage-gated Ca 21 current. Using BaCl 2 (30 mM) as the charge carrier, cells were clamped at 2100 mV and then stepped up in a pulse to 120 mV. (B) Calcium current was not observed in another HN2 clone (HN2V32) transfected with a heterologous gene harbored in the vector pCMV6c. (C,D) Voltage-gated calcium currents observed in HN2A22 and HN2A12 cells, respectively.

parent HN2 cells did not display any calcium current (Fig. 2a) [1], almost all ATPase II-overexpressing clones display voltage-gated calcium current, which was recorded using Ba 21 as the charge carrier (Fig. 2). This Ca 21 current was not an artifact of transfection of the vector (pCMV6) used, because another cell line (HN2V32) [3,27] stably expressing the serotonin 1A receptor from its coding sequence placed in the same vector showed virtually no calcium current (Fig. 2b). Such mixed calcium current was also observed in both APTL-overexpressing clones, HN2A22 and HN2A12 (Fig. 2c,d, respectively). Among 123 HN2A12 cells tested all showed mixed Ca 21 current (average current 319.6617.2 pA) and out of eight HN2A22 cells tested all displayed the mixed calcium current (average current 143.869.5 pA). The mean input resistance of the HN2 cells was 5.0860.60 GV (n513),

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which was not significantly different (P50.87) from that of the HN2A12 cells (4.9060.76 GV; n514). Also, mean membrane capacitance of the HN2 cells was 14.9861.4 pF (n514), which was not significantly different (P50.66) from that of the HN2A12 cells (15.6660.84 pF; n526).

3.3. The voltage-gated calcium current was almost completely inhibited by CdCl2 and significantly attenuated in the presence of v -conotoxin The undifferentiated HN2A12 and HN2A22 cells showed considerable variability in both intensity of the calcium current and also in the extent of inhibition afforded by v-conotoxin. On average 38.566.9% inhibition (n511) of the barium current was observed with 5 mM v-conotoxin (GVIA) (Fig. 3). Nonetheless, virtually

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all of this voltage-gated current was due to calcium channels because an almost complete inhibition was observed in the presence of 100 mM CdCl 2 (Fig. 3a–c).

3.4. Western blotting showed increased expression of the N-type calcium channel alpha 1 B subunit in HN2 A12 and HN2 A22 cells The HN2 cells showed a very faint expression of the 210- and 230-kDa isoforms of the a 1B subunit of the N-type protein channel complex (Fig. 4, lane 1). In contrast, both the ATPase II-overexpressing clones, HN2A12 and HN2A22, showed dramatically (eightfold) increased expression of the immunoreactive 210- and 230kDa protein bands (Fig. 4, lanes 2 and 3). Although the HN2 cells showed a faint expression of the a 1B subunit,

Fig. 3. The voltage-gated Ca 21 current was almost completely inhibited by CdCl 2 and partially eliminated in the presence of v-conotoxin (v-CTX). (A) A typical profile of voltage-gated barium current in undifferentiated HN2A12 cells, which shows almost complete inhibition in the presence of 100 mM CdCl 2 and 38.5% inhibition in the presence of 5 mM v-conotoxin (v-CTX). (B) An individual current recording showing the v-conotoxin and CdCl 2 mediated inhibition of voltage-gated barium current. (C) Average inhibition of voltage-gated calcium current observed in the presence of v-CTX and CdCl 2 . The number shown within each bar indicates the number of cells used for each inhibitor. The CTX-evoked inhibition of current was significant with a P value ,0.05 (paired t-test was used).

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Fig. 4. Increased expression of the a 1B subunit of the N-type calcium channels in the HN2A12 and HN2A22 cells. Samples were resolved by 7–16% gradient SDS-PAGE. Equal protein loading (150 mg) in all lanes was confirmed by reprobing the blot with an antibody to the extracellular receptor-activated kinases 2 (ERK2) (middle panel). Peak areas of immunostained a 1B bands normalized to ERK2 band intensities are presented in the lower panel.

none of our patch-clamp analyses (in the presence of 30 mM Ba 21 ) revealed any calcium current in these cells.

4. 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 Nethylmaleimide (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 internalization of NBD-PS) showed a sharper time-dependent decrease in the Jurkat cells. In addition, as mentioned earlier, a yeast strain mutant in the Drs2 gene, which is homologous to the mammalian ATPase II gene, showed impaired NBD-PS internalization [28]. A recent corroborative finding has shown that deletion of the P-type ATPases, Dnf1p and Dnf2p and Drs2p, which are homologous to the mammalian ATPase II protein causes virtually complete elimination of NBD-PS translocation across yeast membrane [22]. Although such reports indicate that ATPase II

is an aminophospholipid translocase, other studies have argued against this hypothesis [9,26]. Earlier research has demonstrated the presence of higher levels of ATPase II mRNA in the brain and skeletal muscles [13,14,21]. In the brain, the hippocampal neurons show high levels of ATPase II mRNA [14]. Based on such data, we were interested to study the role of ATPase II by overexpressing this protein in hippocampal neuron-derived HN2 cells. This report is focused on the unexpected observation that the overexpression of murine ATPase II resulted in the appearance of mixed, voltage-gated calcium currents in the otherwise calcium current-deficient HN2 cells. This induction of calcium channel expression was not a non-specific effect of the parent vector (pCMV6) that was used to express the ATPase II cDNA, because another cell line (HN2V32) [3,27] expressing a heterologous plasma membrane protein (the serotonin 1A receptor) from the same vector (pCMV6) did not show any calcium current (Fig. 2B) [1]. Such observations bring forth the possibility that ATPases might play an additional role by regulating the expression of other important proteins. This could be the result of stimulation of a signaling cascade by the released phosphate ion, which could then stimulate protein phosphorylation [16,23,24] that is important for the APTL activity. A second mechanistic possibility could be that the ATPase II molecule directly interacts with other proteins that regulate the expression or activity of the calcium channels. It has been shown that extracellular as well as intracellular calcium causes inhibition of both the ATPase and phospholipid-translocase activity of APTL [31]. So, the Ca 21 -sensitive APTL activity of ATPase II could undergo a feedback regulation through increased expression of calcium channels that enhance voltage-gated Ca 21 entry into the cytosol. Either possibility would allow for the elimination of the effect of ATPase II on calcium channels through the down regulation of ATPase II expression. Our future studies will test these possibilities by achieving expression of the ATPase II cDNA and its antisense sequence from an inducible promoter in order to analyze interactions between ATPase II and the expression system of the calcium channels. Both ATPase II-overexpressing clones, HN2A12 and HN2A22, showed dramatically increased expression of the immunoreactive 210- and 230-kDa protein bands (Fig. 4). Although the HN2 cells showed low expression of the a 1B subunit, none of our patch-clamp analyses (in the presence of 30 mM Ba 21 ) revealed any calcium current in these cells. Thus the appearance of Ca 21 current in the HN2A12 and HN2A22 cells could be mostly due to increased gene expression or mRNA stabilization along with some contribution from protein–protein interactions between the calcium channel complex and ATPase II. Collectively, our studies present a novel observation, which might provide valuable insight into the intracellular interactions of crucial functional proteins.

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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, a 1B 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 the NSF Alliance for Minority Participation. This project was supported by the NIH grants CA77803-02 (to P.B.) and ES11022 (to A.W.).

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