©2004 FASEB
The FASEB Journal express article 10.1096/fj.04-2659fje. Published online October 19, 2004.
Alzheimer’s amyloid peptides mediate hypoxic up-regulation of L-type Ca2+ channels Jason L. Scragg,* Ian M. Fearon,† John P. Boyle,* Stephen G. Ball,* Gyula Varadi,‡ and Chris Peers* *Institute for Cardiovascular Research, The University of Leeds, Leeds LS2 9JT, U.K.; † Department of Biology, McMaster University, Hamilton, ON L8S 4K1, Canada; ‡Departments of Surgery and Cell Biology, Neurobiology and Anatomy, University of Cincinnati College of Medicine, Cincinnati, OH 45267-0558, USA Corresponding author: Chris Peers, Institute for Cardiovascular Research, The University of Leeds LS2 9JT, United Kingdom. E-mail:
[email protected] ABSTRACT We examined the effects of chronic hypoxia on recombinant human L-type Ca2+ channel α1C subunits stably expressed in HEK 293 cells, using whole-cell patch-clamp recordings. Current density was dramatically increased following 24 h exposure to chronic hypoxia (CH), and membrane channel protein levels were enhanced. CH also increased the levels of Alzheimer’s amyloid β peptides (AβPs), determined immunocytochemically. Pharmacological prevention of AβP production (via exposure to inhibitors of secretase enzymes that are required to cleave AβP from its precursor protein) prevented hypoxic augmentation of currents, as did inhibition of vesicular trafficking with bafilomycin A1. The enhancing effect of AβPs or CH were abolished following incubation with the monoclonal 3D6 antibody, raised against the extracellular N´ terminus of AβP. Immunolocalization and immunoprecipitation studies provided compelling evidence that AβPs physically associated with the α1C subunit, and this association was promoted by hypoxia. These data suggest an important role for AβPs in mediating the increase in Ca2+ channel activity following CH and show that AβPs act post-transcriptionally to promote α1C subunit insertion into (and/or retention within) the plasma membrane. Such an action will likely contribute to the Ca2+ dyshomeostasis of Alzheimer’s disease and may contribute to the mechanisms underlying the known increased incidence of this neurodegenerative disease following hypoxic episodes. Key Words: chronic hypoxia • Alzheimer’s disease • ion channel
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on channel regulation by O2 tension is a physiologically important and widespread phenomenon (1). Acute hypoxia modulates activity of specific plasmalemmal ion channels thereby controlling cellular excitability (2–4) and consequent functions such as contractility or neurosecretion (5–7). By contrast, chronic hypoxia acts at the transcriptional level, altering expression of numerous proteins including ion channels (8–11). Whether chronic hypoxia (CH) modifies post-transcriptional events has yet to be investigated in depth, but is an important
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question, since hypoxic remodeling of ion channel expression can have deleterious consequences (e.g., (12)). Chronic hypoxia can arise from cardiorespiratory diseases that result in poor arterial blood oxygenation. Such conditions predispose individuals to dementias, of which the most common is Alzheimer’s disease (AD). For example, in patients who have previously suffered ischemic episodes as a consequence of stroke or arrhythmia, the incidence of AD is significantly increased (13–15). A defining feature of AD is the appearance of fibrillar deposits of amyloid β peptides (AβPs; reviewed by (16)). AβPs are neurotoxic, commonly 40–42 amino acids in length, and are cleavage products of amyloid precursor protein (APP; (17, 18)). APP is one of only a few gene products whose expression is increased following cerebral ischemia (19, 20). Because the major cleavage product of APP, soluble APPα (sAPPα), is neuroprotective (16), increased expression of APP may be a defense mechanism against ischemia. However, increased APP levels also provide increased substrate for the formation of neurotoxic AβPs and, indeed, AβP production is increased following ischemia (21, 22), a finding consistent with the clinical association of prolonged hypoxic episodes and AD. The mechanisms underlying the neurodegenerative properties of AβPs, while complex, involve disruption of intracellular Ca2+ homeostasis (16, 23). The major aspect of Ca2+ homeostasis that is disturbed by AβPs is the carefully controlled entry of Ca2+ via voltage-gated Ca2+ channels. We have shown that AβPs selectively up-regulated native L-type Ca2+ currents in PC12 cells (24), while other voltage-gated Ca2+ channels were unaffected. Importantly, this effect was also seen when cells were exposed to chronic hypoxia, and our data indicated that hypoxic upregulation of these channels required AβP formation (11). This effect of hypoxia on native Ltype Ca2+ channels was prevented by inhibition of the transcriptional regulator NF-κB (11). In the present study, we employ a recombinant expression system to investigate the effects of hypoxia on human L-type Ca2+ channel α1C subunits, thereby avoiding transcriptional regulation of channel expression by hypoxia. Our results dramatically show that hypoxia leads to channel up-regulation via post-transcriptional mechanisms that clearly require increased AβP formation. MATERIALS AND METHODS All experiments were conducted in HEK 293 cells stably expressing the human cardiac L-type Ca2+ channel α1C subunit (hHT isoform) alone (i.e., in the absence of auxiliary subunits) using the pcDNA3.1 mammalian expression vector (it is noteworthy that we have also obtained identical preliminary results using the same α1C subunit stably transfected into HEK 293 cells using the pREP9a vector). Cells were maintained in Dulbecco’s Modified Eagle’s Medium containing sodium pyruvate and pyroxidine, but without L-glutamine (Gibco, Paisley, United Kingdom), supplemented with 9% (v/v) fetal calf serum (Helena BioSciences Europe, Sunderland, United Kingdom) and 500 mg l–1 G-418 (Gibco) at 37°C in a humidified atmosphere of 95% air and 5% CO2. Cells were harvested from their culture flasks by trypsinization and plated onto poly-D-lysine-coated Coverslips 24–48 h before use in electrophysiological studies. Cells exposed to chronic hypoxia were treated identically, except that for 24 h prior to experiments they were transferred to a humidified incubator equilibrated with 2.5% O2, 5% CO2, and 92.5% N2. Following this period in chronic hypoxia, cells were exposed to room air for no longer than 1 h before experimentation.
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Electrophysiology Coverslip fragments with attached cells were transferred to a continually perfused recording chamber (perfusion rate 2–4 ml.min–1, volume 80 µl) and whole-cell patch-clamp recordings were made using patch pipettes of resistance 4–7 MΩ. Cells were perfused with a solution containing (in mM): NaCl, 95; CsCl, 5; MgCl2, 0.6; BaCl2, 20; HEPES, 5; D-glucose, 10; TEACl 20 (pH adjusted to 7.4 with NaOH, 21-24°C). Patch electrodes were filled with a solution containing (in mM): CsCl, 120; TEA-Cl, 20; MgCl2, 2; EGTA, 10; HEPES, 10; ATP, 2 (pH adjusted to 7.2 with CsOH). Cells were voltage clamped at –80 mV and whole cell capacitance determined from analog compensation. Series resistance compensation of 70–90% was applied. To evoke whole-cell Ca2+ currents, cells were depolarized either with 200 ms voltage ramps (from –100 mV to +100 mV) at a frequency of 0.1 Hz, or with step depolarizations (100 ms duration, 10 mV increments, from –100 mV to +60 mV). Leak-subtraction was applied as described previously (25). All evoked currents were filtered at 1 kHz, digitized at 2 kHz, and stored on computer for later analysis. All voltage-clamp and analysis protocols were performed with the use of an Axopatch 200A amplifier/Digidata 1200 interface (Clampex software, pCLAMP 6.0.3, Axon Instruments Inc., Foster City, CA 94404, USA). Results are presented as means ± SEM, and statistical analysis was performed using unpaired Student’s t tests. Immunohistochemistry Cells were grown as above, harvested in phosphate-buffered saline (PBS) without Ca2+ or Mg2+ and subcultured on glass Coverslips at a seeding density of 3 × 104 cells/ml. After four days in culture cells were washed in PBS, then fixed in 4% paraformaldehyde. Visualizing α1C channels via immunohistochemistry required that the cells were permeabilized with 0.02% Triton-X100 in PBS supplemented with 10% goat serum while for AβP immunohistochemistry, Coverslips were incubated in PBS containing 10% goat serum. The cells were incubated (4°C, overnight) in the presence of either a polyclonal antibody raised against residues 848–865 of the rat α1C subunit (Alamone Labs, Jerusalem, Israel) or 3D6, a monoclonal antibody raised against AβP1-5 (provided by Elan Pharmaceuticals, San Diego, CA), in PBS with 1% goat serum. Antibody binding was visualized using a Cy3-conjugated anti-mouse or anti-rabbit antibody. Coverslips were mounted on slides using Vectashield (Vector Laboratories Ltd., Burlingame, CA) and examined using either a Zeiss epifluorescence microscope or a Zeiss laser scanning confocal microscope (LSM 510). Cells for colocalization studies were treated as above, excepting that all cells were permeabilized, and overnight incubation at 4°C was performed in the presence of both anti-α1C and 3D6. Antibody binding was visualized using anti-mouse conjugated Alexa Fluor 555 and anti-rabbit conjugated Alexa Fluor 488 (Molecular Probes). Cells were examined on a Zeiss LSM 510, and fluorophores excited using helium/neon and argon lasers and the composite images and scan lines produced using Carl Zeiss AIM LSM 510 software. Immunoprecipitation and Western blots For coimmunoprecipitation or immunoblotting, cells were grown as above to 80–90% confluence in 75 cm2 or 225 cm2 flasks and maintained under normoxic or hypoxic conditions, as above. For Western blotting of whole cell extracts, cells were washed in ice-cold PBS and lysed “in situ” with 1 ml mammalian protein extraction reagent (M-PER, Pierce) containing Complete Page 3 of 19 (page number not for citation purposes)
miniprotease inhibitor tablets (Roche Bioscience, Palo Alto, CA) for 30 min at room temperature. Protein levels in lysates were assessed using the method of Bradford (26). For immunoblotting of membrane-enriched extracts, cells were washed in ice-cold PBS, harvested in 10 ml ice-cold PBS and briefly centrifuged at 600 g. Cell pellets were homogenized in 1 ml icecold buffer (50 mM Tris-HCl, 140 mM KCl, 1 mM EGTA and 1 mM MgCl2, pH 7.4 supplemented with a Complete miniprotease inhibitor tablet) and centrifuged at 600 g for 2 min at 4°C. The supernatants were removed and centrifuged at ~16,000 g for 40 min at 4°C, and pellets were solubilized in 100 µl mammalian protein extraction reagent containing a Complete miniprotease inhibitor tablet. Cell proteins (typically 10 µg protein per lane) were separated on 7.5%, 0.75-mm thick polyacrylamide SDS gels and electrophoretically transferred to PVDF membranes (Bio-Rad Laboratories, Hercules, CA). Blots were probed with the same anti-α1C antibody as used for immunohistochemistry (Alamone Labs), and bands were visualized using an enhanced chemiluminescence detection system and hyperfilm ECL (Amersham-Pharmacia Biotech Inc., Piscataway, NJ). For coimmunoprecipitation experiments, cell pellets were solubilized in 500 µl ice-cold buffer (5 mM Tris-HCl, 500 mM NaCl, 1.5% v/v Triton X-100, pH 7.4) containing a Complete miniprotease inhibitor tablet. This solution was centrifuged at 3,000 g for 4 min and the supernatant transferred to fresh tubes. 5 µg antibody (either anti-α1C or 3D6) was added to the supernatant and incubated at 4°C for 1 h before the addition of 50 µl protein G or protein Aconjugated agarose beads. The mixture was then incubated, with gentle tumbling, at 4°C for at least 24 h. The beads were pelleted at 5,000 g for 5 min and washed twice with 1 ml ice-cold buffer (5 mM Tris-HCl, 20 mM NaCl, 0.5% v/v Triton X-100, pH 7.4) containing a Complete miniprotease inhibitor tablet. Pelleted immunoprecipitates were taken up into 50 µl sample buffer (62.5 mM Tris-HCl, 10% v/v glycerol, 0.2% w/v SDS, 5% v/v β-mercaptoethanol, 0.02% w/v bromophenol blue, pH 6.8) and, where the capture antibody was anti-α1C, heated in boiling water for 2 min and loaded, alongside protein molecular weight markers onto Tris/tricine gels for electrophoresis. Where the capture antibody was 3D6, samples were not boiled, as this caused the appearance of multiple bands and smearing on the gel, but were left to stand at room temperature, with occasional gentle mixing, for 10–15 min before loading onto 7.5% polyacrylamide gels for electrophoresis. Gels were run at a constant current of 30 mA, and proteins were transferred to PVDF membranes overnight. The blots were blocked in PBS containing 0.2% Tween and 5% milk protein for 1 h and then incubated with continuous rolling, at room temperature for 3 h, in the same solution containing either 3D6 (where the capture antibody was anti α1C) or anti-α1C (where the capture antibody was 3D6). Blots were washed in PBS/Tween (6 × 5 min) and incubated for a further 1 h in the presence of either donkey anti-rabbit or donkey anti-mouse antibody conjugated to horseradish peroxidase (Amersham Biosciences). After washing (PBS/Tween, 6 × 5min) bands were labeled using ECL (Amersham Biosciences) and bands visualized on Hyperfilm ECL (Amersham Biosciences). RESULTS Twenty-four hours of chronic hypoxia (CH; 2.5% O2) significantly (P
