Depletion of 14-3-3γ reduces the surface expression of Transient Receptor Potential Melastatin 4b (TRPM4b) Channels and attenuates TRPM4b-mediated glutamate-induced neuronal cell death
© Cho et al. 2014
Received: 7 April 2014
Accepted: 14 July 2014
Published: 22 July 2014
TRPM4 channels are Ca2+-activated nonselective cation channels which are deeply involved in physiological and pathological conditions. However, their trafficking mechanism and binding partners are still elusive.
We have found the 14-3-3γ as a binding partner for TRPM4b using its N-terminal fragment from the yeast-two hybrid screening. Ser88 at the N-terminus of TRPM4b is critical for 14-3-3γ binding by showing GST pull-down and co-immunoprecipitation. Heterologous overexpression of 14-3-3γ in HEK293T cells increased TRPM4b expression on the plasma membrane which was measured by whole-cell recordings and cell surface biotinylation experiment. Surface expression of TRPM4b was greatly reduced by short hairpin RNA (shRNA) against 14-3-3γ. Next, endogenous TRPM4b-mediated currents were electrophysiologically characterized by application of glutamate and 9-phenanthrol, a TRPM4b specific antagonist in HT-22 cells which originated from mouse hippocampal neurons. Glutamate-induced TRPM4b currents were significantly attenuated by shRNAs against 14-3-3γ or TRPM4b in these cells. Finally, glutamate-induced cell death was greatly prevented by treatment of 9-phenanthrol or 14-3-3γ shRNA.
These results showed that the cell surface expression of TRPM4 channels is mediated by 14-3-3γ binding, and the specific inhibition of this trafficking process can be a potential therapeutic target for glutamate-induced neuronal cell death.
KeywordsTRPM4b 14-3-3 Non-selective cation channels 9-phenanthrol HT-22 Protein-protein interaction Surface expression Hippocampal neurons Calcium activated cation channels MTT assay
The calcium ion (Ca2+) is the most abundant signaling molecule in regulating physiological and cellular functions []. Therefore, it is natural that Ca2+-permeable membrane proteins have been vigorously studied to understand their dynamic actions: voltage-gated Ca2+ channels, Ca2+-permeable TRP (Transient Receptor Potential) channels, ligand-gated Ca2+-permeable receptors (e.g., ionotrophic glutamate receptors, purinergic receptors and nicotinic acetylcholine receptors), and intracellular receptors such as ryanodine and IP3 receptors [-]. In addition, Ca2+-activated K+ channels (BK, IK, and SK), and Ca2+-activated Cl− channels (Bestrophins and Anoctamins) are getting more attentions recently [,].
Although Ca2+-activated Na+ channels have yet to be found, Ca2+-permeable non-selective cation channels are numerous (e.g., NMDA receptors and TRPC channels). In contrast, Ca2+-impermeable non-selective cation channels are only a few: AMPA receptors (except for AMPA receptors with calcium permeable unedited GluR2 subunit or without GluR2 subunit) and TRPM4 and TRPM5 [-]. TRPM4 and TRPM5 have been identified as Ca2+-activated, Ca2+-impermeable monovalent cation channels whose activities have been first reported more than three decades ago [-]. They conduct Na+ and K+ according to the concentration gradient of individual ion when activated by membrane depolarization and an increase in intracellular calcium ([Ca2+]i) [-]. They belong to a subgroup (melastatin-related TRPM) of TRP channels with six transmembrane domains which forms a functional channel as a tetramer [,].
TRPM4 channels are expressed in various tissues including brain [-]. Mutations in TRPM4 gene and their consequential dysfunction have been linked to cardiac diseases [-]. The increased level of TRPM4 channels has been reported in vascular endothelium following hypoxia/ischemia, in the endothelial cells of capillary vessels following spinal cord injury [,]. In the brain, expression and/or channel activities of TRPM4 have been detected in hippocampus, cerebellar Purkinje cells, preBӧtzinger complex in the brainstem, magnocellular cells in supraoptic and paraventricular nuclei, and substantia nigra pars compacta [-]. Recently, TRPM4 has been shown to be important in neuronal cell death of experimental autoimmune encephalomyelitis and human multiple sclerosis tissues []. Therefore, understanding trafficking mechanism of TRPM4 channels to the plasma membrane may open the therapeutic window to intervene the underlying TRPM4-related diseases.
Results and discussions
Identification of 14-3-3γ as a binding partner of TRPM4b
Ser88 at the N-terminus of TRPM4b is critical for 14-3-3γ binding
Surface expression of TRPM4b is mediated by 14-3-3γ
Next, we measured TRPM4b-mediated whole-cell currents elicited by 1 μM ionomycin from the HEK293T cells overexpressing GFP-TRPM4b with or without mCherry-14-3-3γ using the voltage-ramp from −100 to +100 mV []. Currents were measured at 5 min after ionomycin application and ionomycin-activated currents were calculated by subtracting the initial currents from them (Figure 3C and D). The current density from cells overexpressing TRPM4b and 14-3-3γ were significantly larger than the ones from cells overexpressing TRPM4b alone. In addition, the currents from cells overexpressing TRPM4b-S88A mutants were negligible and similar to the ones from non-transfected cells. Alternatively, using the pipette solution including 30 μM [Ca2+]i the current density from the cells overexpressing both TRPM4b and 14-3-3γ were more than two-folds larger than those from the cells overexpressing TRPM4b alone (see Additional file 2: Figures S2A and B).
Deficiency of 14-3-3γ reduces the surface expression of TRPM4b in HEK293T cells
Glutamate-induced activation of endogenous TRPM4b in neuronal cells from hippocampus
Recently, the deficiency of TRPM4 has been shown to be neuroprotective from glutamate-induced cell death in experimental autoimmune encephalomyelitis, a mouse model of human multiple sclerosis []. This report leads us to hypothesize that interference of TRPM4b function via blocking its trafficking to the plasma membrane with 14-3-3γ shRNA may have a protective effect from glutamate neurotoxicity. To test this hypothesis we first characterized TRPM4b-mediated currents in HT-22 cells, originated from mouse hippocampal neurons, which have been frequently used as an in vitro model for glutamate-induced neurotoxicity [].
We also examined the endogenous expression of TRPM4b in HT-22 cells by quantitative RT-PCR. As shown in Additional file 3: Figure S3C, TRPM4b expression in HT-22 cells was comparable to its expression in cultured hippocampal neurons. To rule out the possibility of glutamate-induced current activation through other than TRPM4b channels, we designed specific shRNAs against TRPM4b and found two functional shRNA constructs (see Additional file 3: Figure S3D). Glutamate-elicited currents was indeed failed to be activated in HT-22 cells transfected with a specific TRPM4b shRNA (Figure 5C and D). In summary, this data clearly showed that HT-22 cells have functional TRPM4b channels which can be elicited by glutamate.
Now, to examine the efficient interference of TRPM4b trafficking with 14-3-3γ shRNA, we measured glutamate-induced TRPM4b currents in HT-22 cells transfected with either 14-3-3γ shRNA or Sc shRNA. In contrast to the current amplitude of glutamate-induced TRPM4b channels in the cells transfected with Sc shRNA, the one in the cells transfected with 14-3-3γ shRNA was drastically reduced even 20 min after glutamate application (P < 0.001; Figure 5C and D).
Lastly, we asked whether inhibition of activity and trafficking of TRPM4b may prevent the neuronal cells from glutamate-induced cell death. First, we tested the effect of 9-phenanthrol on the glutamate-induced cell death using the MTT assay since 9-phenanthrol blocked TRPM4b channels effectively in whole-cell recordings. The great number of HT-22 cells was killed after glutamate (5 mM) treatment (69.57 ± 0.92% (n = 24); Figure 5E). In comparison, the cell death was dramatically reduced by 9-phenanthrol in a dose-dependent manner and no significant difference in control cells (without glutamate treatment) was detected (Figure 5E). We also examined the effect of 14-3-3γ shRNA on the cell viability in glutamate-treated HT-22 cells. Compared to the HT-22 cells transfected with Sc shRNA, knockdowns of 14-3-3γ and TRPM4b increased the cell survival (136.1 ± 3.6% (n = 24) and 129.8 ± 4.6% (n = 6), respectively; Figure 5F).
14-3-3γ-mediated TRPM4b trafficking mechanism provides a potential therapeutic target
14-3-3 proteins are one of most important ‘hub’ proteins involved in diverse cellular processes in all eukaryotes []. Their isoforms are broadly expressed in nervous systems and involved in many physiological conditions (such as hippocampal long-term potentiation) as well as neurodegenerative and neuropsychiatric diseases [-]. 14-3-3 proteins bind to a lot of membrane proteins including ion channels such as TASK-1 and TASK-3, TRESK, KATP channels, hERG/IKr channels, NaV1.5, CaV2.2, GABAB receptors, neuronal nicotinic α4β2 receptors, NMDA receptor subunit NR2C, kainate receptor subunit GluK2 and GluK5 [-]. Among isoforms, 14-3-3γ has been shown to interact with a large-conductance Ca2+-activated K+ (BK) channels and increases their surface expression as shown in this study []. In general, 14-3-3 proteins bind to (mostly) phosphorylated target proteins at the specific site(s) and function as a dimer []. A previous study showed that PKCδ activation increased the surface expression of TRPM4 channels in vascular smooth muscle cells []; however, two putative PKC phosphorylation sites are localized at the C-terminus of TRPM4 channels []. Since 14-3-3γ binds to the N-terminus of TRPM4b in this study, kinase(s) other than PKC may be involved in 14-3-3γ-mediated trafficking of TRPM4b channels.
A recent report showed that the glutamate-induced neuronal cell death was absent in TRPM4 null mice [] and our results also clearly showed that inhibition of TRPM4b function either by a specific inhibitor or interfering trafficking of TRPM4b channels to the plasma membrane via 14-3-3γ shRNA blocked glutamate-induced cell death (Figure 5). Therefore, TRPM4b channels can be new therapeutic targets. R18 and difopein (dimeric fourteen-three-three peptide inhibitor), peptide inhibitors against 14-3-3 proteins, are available, however, specific inhibitors against each 14-3-3 isoforms have not been developed for the therapeutic purpose []. Instead, a gene therapy using shRNA against 14-3-3γ in a cell type-specific manner could be one promising way to ameliorate devastating neurological diseases. Especially, 14-3-3γ has been deeply involved in neurological diseases; inflammatory joint diseases [], Alzheimer’s diseases [], Parkinson’s disease [], multiple sclerosis [], and Creutzfeldt-Jakob disease [,]. Whereas TRPM4b channels have not been studied in these diseases, 14-3-3γ-mediated TRPM4b trafficking might be involved in the neuronal cell death in these diseases.
TRPM4b channels are Ca2+-activated, Ca2+-impermeable monovalent cation channels which are expressed in various neuronal tissues and involved in diverse physiological conditions. We have identified 14-3-3γ as a trafficking molecule for TRPM4b and Ser88 residue at the N-terminus of TRPM4b is critical for 14-3-3γ binding. Heterologous overexpression of 14-3-3γ showed the increased TRPM4b expression on the plasma membrane, in contrast, the surface expression of TRPM4b was greatly reduced by the knockdown of endogenous 14-3-3γ. Glutamate-induced TRPM4b currents and glutamate-induced neurotoxicity in HT-22 cells were attenuated by a TRPM4b specific antagonist and 14-3-3γ shRNA. Taken together, these results suggested that specific inhibition of 14-3-3γ-mediated trafficking of TRPM4b channels provide a new therapeutic target against neuronal cell death.
Ionomycin, 9-phenanthrol, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and all other chemicals were purchased from Sigma Aldrich.
Plasmid and shRNA construction
The cDNA encoding for full-length human TRPM4b (Genebank accession no. NM_017636), mouse TRPM4b (Genebank accession no. NM_175130), and human 14-3-3γ (Genebank accession no. NM_012479) were obtained by the PCR-based gateway cloning method (Invitrogen). The cDNAs of 14-3-3 isoforms were kindly provided by Dr. Dukryong Kim (Gyeoungsang National University). TRPM4b-N174 was generated from the full-length cDNA, and its phosphorylation mutants (T19A, T42A and S88A) were made by EZchange site directed mutagenesis kit (Enzynomics). All constructs were cloned into several vectors, including pDEST-EGFP-C, pDEST-mCherry-C, pDEST-FLAG-C, pDEST-HA-C by gateway cloning. The target sequence of shRNAs as follows: human 14-3-3γ 5’-ACGAGGACTCCTACAAGGAC-3’, mouse 14-3-3γ 5’-GGACAACTACCTGATCAAGAA-3’, mouse TRPM4b 5’-GCTGGATCCCTAAGATCTTCA-3’.
Yeast two-hybrid screening
The N-termini of human TRPM4b (N174) was ligated into pGBKT7 encoding for the GAL4 DNA binding domain (BD) and human cDNA library was cloned into pGADT7 encoding for the activation domain (AD). BD/TRPM4b-N174 and AD/cDNA library were co-transformed into the yeast strain AH109 in order to find protein-protein interactions with TRPM4b-N174. AH109 was unable to synthesize histidine but AD/14-3-3γ was found to grow on His minimal medium by permitting histidine biosynthesis.
Cell culture and transfection
HEK293T and HT-22 cells were purchased from the Korean Cell Line Bank (Seoul National University) and cultured in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen), 100 units per ml penicillin-streptomycin (Invitrogen) at 37°C in a humidified atmosphere of 95% O2 and 5% CO2. Transfection of expression vectors was performed with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol.
Co-immunoprecipitation and immunoblotting
For co-immunoprecipitation, whole-cell lysates were mixed at 4°C with 1 μg/ml anti-FLAG (M2, sigma) antibodies in lysis buffer (in mM, 50 Tris–HCl (pH 7.4), 150 NaCl, 5 EDTA, 1 PMSF and 1% NP-40) containing a protease-inhibitor cocktail (Roche). Immune complexes were incubated by binding to mixed protein A/G PLUS-Agarose (Santa Cruz Biotechnology) for 1 hr, and washed four times with lysis buffer. For immunoblotting, Proteins were separated by 10% SDS-PAGE gel and blotted onto PVDF membranes. The blots were blocked with 5% skim-milk in TBST at room temperature for 20 min and incubated for overnight at 4°C with anti-GFP antibody (1:500, Santa Cruz Biotechnology), anti-FLAG (1:1000, Sigma) or anti-HA antibody (1:500, Roche). Blots were then washed and incubated with horse-radish peroxidase conjugated goat anti-mouse IgG, followed by washing and detection of immunoreactivity with the enhanced chemiluminescence (Amersham Biosciences).
GST pull-down experiment
To produce GST-14-3-3γ (14-3-3γ fused with Glutathione-S-transferase) construct, the full-length human 14-3-3γ was cloned into the pDEST15 vector (Invitrogen) via the gateway cloning system. GST-14-3-3γ proteins were expressed in bacteria and purified according to the manufacturer’s protocol (Amersham Biosciences). Cell lysates obtained from wildtype or mutants of TRPM4b-N174 transfected cells were incubated with GST-14-3-3γ purified from bacteria for 2 hrs, and then incubated with glutathione-Sepharose 4B beads (Amersham Biosciences). After 2 hrs incubation at 4°C, the beads were washed four times with ice cold lysis buffer. Bound proteins were eluted with SDS sample buffer, separated on 10% SDS–PAGE gels, and analyzed by Western blot.
Cell Surface biotinylation
For surface biotinylation, the cell membrane proteins of GFP-TRPM4b transfected HEK293T cells were biotinylated with PBS containing Sulfo-NHS-SS-biotin (Pierce) at 4°C for 30 min. After biotinylation, cells were washed with quenching buffer (100 mM glycine in PBS) to remove excess biotin and washed three times with PBS. The cells were then lysed and incubated with high capacity NeutrAvidin-Agarose Resin (Thermo Science). After three washes with lysis buffer, bound proteins are eluted by SDS sample buffer and subjected to Western blot analysis.
Bimolecular fluorescence complementation (BIFC) experiment
TRPM4b and 14-3-3γ were cloned into bimolecular fluorescence complement (pBiFC)-VN173 and pBIFC-VC173 vectors. HEK293T cells were co-transfected with cloned BiFC vectors and transfected with DsRed-Mem for the detection of plasma membrane. These cells were fixed with 4% paraformaldehyde for 20 min at room temperature and mounted with Dako fluorescence mounting medium. Venus fluorescence signals were observed by confocal microscopy (Nikon A1).
Cell surface expression of TRPM4b was determined using a confocal microscope. HEK293T cells were plated onto glass coverslips and co-transfected with GFP-TRPM4b and DsRed-Mem. The cells were cultured for additional 24 hrs, washed twice with PBS, and directly observed under a confocal microscope. Pearson’s correlation coefficients were calculated from the co-localization signals at the edge region of cells using the Nikon A1 confocal software.
The standard solution for bath and pipette contained, in mM: 1 MgCl2, 5.5 glucose, and 10 HEPES. Na-gluconate (bath, 145 and pipette, 120) was used. Pipette solution contained 10 mM EGTA and pH was adjusted with NaOH to 7.2, while bath solution contained 2 mM CaCl2 and adjusted to pH 7.35. K+ was not included in either recording or pipette solution to eliminate the Ca2+-activated K+ channels which might be endogenously expressed in either HT293T or HT-22 cells. In a few initial experiments, tetraethylammonium (TEA, 2 mM) was added to the bath solution, however, it did not show any difference from the data with no TEA. Therefore, the data with and without TEA were pooled. Patch pipettes were made from borosilicate glass capillaries (Warner Instruments, Inc.). Whole-cell currents were recorded using a patch clamp amplifier (Axopatch 200B, Axon Instrument, Inc.). The current–voltage relations were measured by applying ramp pulses (from – 100 mV to +100 mV during 400 ms) from a holding potential of 0 mV at room temperature. The sampling interval was 200 μs and the filter setting was 2 KHz. Currents were analyzed with Clampfit software (Axon instruments, Inc.). Ionomycin (1 μM) was used to activate TRPM4b channels overexpressed in HEK293T cells. The current densities were calculated by subtracting the amplitude of initial currents from activated TRPM4b currents and divided by cell capacitance. A TRPM4b inhibitor, 9-phenanthrol (100 μM) was applied when glutamate-induced TRPM4b currents reached to near maximum values (usually 10 – 20 minutes after glutamate (1 mM) application) to measure the inhibition in HT-22 cells.
Real-time quantitative RT-PCR (qRT-PCR)
Total RNA was isolated from HT-22 cells and primary cultured hippocampal neurons using a RNA purification kit (GeneAll) according to the manufacturer’s instruction. The cDNA was synthesized from 1 μg of total RNA and reverse transcription was performed with SuperScript® VILO™ cDNA Synthesis Kit (Invitrogen). The qRT-PCR was done with a SensiFAST™ Probe Hi-ROX Kit (Bioline) using the TaqMan probe Mm.PT.58.8488376 (IDT). GAPDH was used as an endogenous control. The 2-ΔΔCt method was used to calculate fold changes in gene expression [].
MTT cell viability experiment
Cell viability of HT-22 cells was assessed by MTT assay []. 9-phenanthrol (1, 3 and10 μM) was applied 30 min before incubating in glutamate (5 mM) for 18 hr. After treatment, MTT reagent (5 mg/ml in PBS) was added to the culture wells, incubated for 3 hr, and the samples were lysed with Dimethyl Sulfoxide (DMSO). MTT is reductively converted into a violet formazan derivative, which is quantified photometrically at 570 nm in a well plate reading system (Infinite M200 pro). The mean values from the readings of triplicate wells were taken as one value. The optical density value for the control cultures was considered as 100% viability (control) and viability in other samples is expressed as a percentage of viability in the control culture.
Numerical data are presented as means ± SEM. Error bars in graphs denote the standard error of the mean. The statistical significance of data was assessed by unpaired or paired Student’s t-test. The significance level is displayed as asterisks (*P < 0.05 or **P < 0.01, ***p < 0.001).
(Green Fluorescent Protein)
This work was supported by the National Research Foundation funded by the Korean Ministry of Science, Education and Technology (WCI 2009–003 and 2009–0067148).
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