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. 2014 Aug;17(8):1055-63.
doi: 10.1038/nn.3744. Epub 2014 Jun 22.

Nuclear BK Channels Regulate Gene Expression via the Control of Nuclear Calcium Signaling

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Free PMC article

Nuclear BK Channels Regulate Gene Expression via the Control of Nuclear Calcium Signaling

Boxing Li et al. Nat Neurosci. .
Free PMC article

Erratum in

  • Nat Neurosci. 2014 Dec;17(12):1841

Abstract

Ion channels are essential for the regulation of neuronal functions. The significance of plasma membrane, mitochondrial, endoplasmic reticulum and lysosomal ion channels in the regulation of Ca(2+) is well established. In contrast, surprisingly little is known about the function of ion channels on the nuclear envelope (NE). Here we demonstrate the presence of functional large-conductance, calcium-activated potassium channels (BK channels) on the NE of rodent hippocampal neurons. Functionally, blockade of nuclear BK channels (nBK channels) induces NE-derived Ca(2+) release, nucleoplasmic Ca(2+) elevation and cyclic AMP response element binding protein (CREB)-dependent transcription. More importantly, blockade of nBK channels regulates nuclear Ca(2+)-sensitive gene expression and promotes dendritic arborization in a nuclear Ca(2+)-dependent manner. These results suggest that the nBK channel functions as a molecular link between neuronal activity and nuclear Ca(2+) to convey signals from synapse to nucleus and is a new modulator, operating at the NE, of synaptic activity-dependent neuronal functions.

Figures

Figure 1
Figure 1. BK channels expression on the NE of hippocampal neurons
(a) The confocal immunofluorescence of BK channel and lamin B in cultured hippocampal neurons. Analysis of intensity profiles of the vertical line across the center of the nucleus was shown on the right. Over 85% of the neurons (>100 neurons) have the positive BK channel staining in NE. (b) Immunoelectron microscopy images of BK channel localization in cultured hippocampal neurons (Top). Gold particles signaling BK channels were localized on NE (arrows) and mitochondria (M) (arrowhead) in wild-type mice. Forty-six immunoelectron microscopy images were taken totally. NE–localized gold particles were observed in all the images from the wild-type mice, and there is no signal in Kcnma1–/– mice. Scale bar represents 1 μm. Middle, schematic diagram of the top images. Bottom, an enlargement of a square region indicated in the top images. (c) Left, immunofluorescence of BK channels and lamin B in isolated nuclei. Right, analysis of intensity profiles of the horizontal line across the center of the isolated nuclei. (d) Immunofluorescence of BK-GFP and lamin B. (e) Western blot results of the subcellular localization of the BK channel in the membrane fraction (M), the whole cell lysate (W), and the nuclear fraction (N). (f) Western blot results of the BK channel expression in intact isolated nuclei (1), denuded nuclei (2) and NE (3). (g) Single channel activities of the BK channel in isolated nuclei with 10 μM Ca2+ at different voltage in control group (left), with paxilline treatment (10 μM, middle), and in the nuclei from Kcnma1–/– mice (right) (n = 6). (h) Left, single channel activities recorded in the solutions with 10 μM or 5 μM Ca2+ at 40 mV. Right, BK channel open probability (PO) recorded in the solutions with 10 μM (n = 10 for each group, unpaired t-test, P = 2.30 × 10–14, t18 = 21.71 between control and paxilline) or 5 μM Ca2+ (n = 10 for each group, unpaired t-test, P = 1.80 × 10–8, t18 = 9.55, between control and paxilline) at 40 mV (error bars are mean ± s.d.). (i) I-V curve of the channel activity. For single channel recording, BK channel activity was recorded from 15 of the 20 successful patching of the nuclei from wild-type mice. All the data are from 3 independent cultures from at least 3 litters. All the experiments of af were successfully repeated for at least 3 times. WT, wild-type. Scale bars in a,c,d represent 10 μm. The full-length blots for e and f are presented in Supplementary Figure 7.
Figure 2
Figure 2. nBK channels regulate the nuclear transmembrane potential and nuclear Ca2+ concentration in intact neurons and isolated nuclei
(a) Fluorescence intensity changes of DiOC6(3)-loaded isolated nuclei (insert) upon addition of paxilline. (b) Confocal nuclear calcium imaging with Fluo-4/AM of the mid-nuclear region in hippocampal neurons. (c) The changes of fluorescence intensity in nuclear calcium imaging of intact neurons after indicated treatments (left). Right, summary of the data. One-way ANOVA (n = 15 for each group, P = 4.70 × 10–7, F4,70 = 11.15) and post hoc test. (d) Fluorescence intensity changes in the nuclei of digitonin-permeabilized neurons after paxilline treatment. (e) Paxilline-induced fluorescence intensity changes in intact neurons from wild-type and Kcnma1–/– mice (n = 8 for each group, unpaired t-test, P = 2.56 × 10–5, t14 = 6.14, points with error bars represent the mean ± s.e.m.). Paxilline-induced changes in Fluo-4/AM (f) or Fluo-4/dextran (g) fluorescence intensity in the isolated nuclei. (h) The changes of fluorescence intensity in isolated nuclei after indicated treatments (left). Right, summary of the data. One-way ANOVA (n = 11, 8, 12, 8, 10, 10, respectively, P = 2.50 × 10–13, F5,53 = 26.55) and post hoc test. (i) The changes of fluorescence intensity in isolated nuclei after knockdown of RyRs (left). Right, summary of the data. One-way ANOVA (n = 5, 6, 6, 5, respectively, P = 0.0016, F3,18 = 7.72) and post hoc test. Fisher's least significant difference test was used for post hoc test in one-way ANOVA. Error bars represent the mean ± s.e.m.. All the data are from 3 independent cultures from at least 3 litters. All the experiments were successfully repeated for at least 3 times. **P < 0.01. WT, wild-type; Pax, paxilline; RR, ruthenium red; TG, thapsigargin; shRNA, RyRs-specific shRNAs; N.S., non-silencing shRNAs. Scale bars in a,b,f,g represent 5 μm.
Figure 3
Figure 3. Blockade of nBK channels induces CREB phosphorylation in isolated nuclei and intact neurons
(a) Immunoblots of isolated functional nuclei after stimulation as indicated. One-way ANOVA (n = 4 for each group; P = 1.75 × 10–5, F2,9 = 46.81 for p-CREB; P = 0.0018, F2,9 = 13.91 for p-CaMKIV; P = 0.87, F2,9 = 0.14 for p-ERK) and post hoc test. (b) Immunofluorescence of CREB phosphorylation. (c) Statistic results of b. One-way ANOVA (N = 5 for each group, P = 3.89 × 10–6, F2,12 = 41.85) and post hoc test. (d,f) Immunoblots of isolated functional nuclei (d) and intact neurons (f) after paxilline treatment. (e,g) Immunofluorescence intensity of CREB phosphorylation in isolated functional nuclei (e) and intact neurons (g) after treatments as indicated. One-way ANOVA (N = 5 for each group; P = 0.0093, F3,16 = 5.40 for data in e; P = 0.00096, F3,16 = 9.08 for data in g) and post hoc test. (h) CaMKIV expression after shRNA transfection. (i) Left, paxilline-induced CREB phosphorylation in the neurons transfected with GFP-tagged plasmids encoding CaMKIV-specific shRNAs or non-silencing shRNAs (N.S.), respectively. Right, summary of the data. One-way ANOVA (N = 5 for each group, P = 0.0021, F3,16 = 7.67) and post hoc test. (j) Paxilline-induced CREB phosphorylation after treatments as indicated. One-way ANOVA (N = 5 for each group, P = 5.64 × 10–6, F4,20 = 15.76) and post hoc test. Fisher's least significant difference test was used for post hoc test in one-way ANOVA. Error bars represent the mean ± s.e.m.. All the data in a are from 3 independent cultures from at least 3 litters. All the other data are from 5 coverslips of cells from 3 independent cultures from at least 3 litters. All the experiments were successfully repeated for at least 3 times. **P < 0.01; n.s., no significant difference. WT, wild-type; N.S., non-silencing shRNAs. Scale bars in b,h represent 10 μm. The full-length blots for a, d, f and h are presented in Supplementary Figure 7.
Figure 4
Figure 4. nBK channels regulate synaptic activity-evoked [Ca2+]nu elevation, CREB phosphorylation and gene expression
(a) Bicuculline-induced changes of fluorescence intensity in nuclear calcium imaging of intact neurons after DMSO or paxilline pretreatment. (b) Left, bicuculline-induced CREB phophorylation in intact neurons with or without paxilline pretreatment. Scale bar represents 20 μm. Right, summary of the data. One-way ANOVA (N = 5 for each group; P = 4.98 × 10–5, F2,12 = 25.29 for wild-type group, P = 3.15 × 10–5, F2,12 = 27.76 for Kcnma1–/– group) and post hoc test. (c) Left, mRNA level of c-fos after treatments as indicated. One-way ANOVA (P = 0.0015, F2,6 = 23.34 for wild-type group; P = 0.90, F2,6 = 0.11 for Kcnma1–/– group) and post hoc test. Right, bicuculline-induced c-fos expression after DMSO or paxilline pretreatment (unpaired t-test, P = 0.0053, t4 = –5.50 for control group; P = 0.0093, t4 = 4.70 for bicuculline group). (d) Left, mRNA level of Npas4 after treatments as indicated. One-way ANOVA (P = 0.00090, F2,6 = 28.02 for wild-type group; P = 0.99, F2,6 = 0.00999 for Kcnma1–/– group) and post hoc test. Right, bicuculline-induced Npas4 expression after DMSO or paxilline pretreatment (unpaired t-test, P = 0.0035, t4 = –6.16 for control group; P = 0.0082, t4 = 4.87 for bicuculline group). (e) Left, mRNA level of Atf3 after treatments as indicated. One-way ANOVA (P = 0.00048, F2,6 = 35.38 for wild-type group; P = 0.90, F2,6 = 0.11 for Kcnma1–/– group) and post hoc test. Right, bicuculline-induced Atf3 expression after DMSO or paxilline pretreatment (unpaired t-test, P = 4.29 × 10–5, t4 = –19.25 for control group; P = 0.0023, t4 = 6.88 for bicuculline group). (f) Left, mRNA level of Btg2 after treatments as indicated. One-way ANOVA (P = 0.018, F2,6 = 8.36 for wild-type group; P = 0.16, F2,6 = 0.16 for Kcnma1–/– group) and post hoc test. Right, bicuculline-induced Btg2 expression after DMSO or paxilline pretreatment (unpaired t-test, P = 0.0076, t4 = –4.98 for control group; P = 0.0057, t4 = 5.40 for bicuculline group). (g) Left, mRNA level of Bcl6 after treatments as indicated. One-way ANOVA (P = 0.0011, F2,6 = 25.61 for wild-type group; P = 0.93, F2,6 = 0.074 for Kcnma1–/– group) and post hoc test. Right, bicuculline-induced Bcl6 expression after DMSO or paxilline pretreatment (unpaired t-test, P = 0.0034, t4 = –6.21 for control group; P = 0.0040, t4 = 5.96 for bicuculline group). (h) Left, mRNA level of Ifi202b after treatments as indicated. One-way ANOVA (P = 0.011, F2,6 = 10.45 for wild-type group; P = 0.93, F2,6 = 0.075 for Kcnma1–/– group) and post hoc test. Right, bicuculline-induced Ifi202b expression after DMSO or paxilline pretreatment (unpaired t-test, P = 0.0094, t4 = –4.69 for control group; P = 0.0030, t4 = 6.44 for bicuculline group). N = 3 independent cultures from at least 3 litters for each group in ch. Fisher's least significant difference test was used for post hoc test in one-way ANOVA. Error bars represent the mean ± s.e.m.. **P < 0.01; n.s., no significant difference. WT, wild-type.
Figure 5
Figure 5. nBK channels regulate dendritic arborization via nuclear Ca2+/CaMKIV signaling
(a) Representative micrographs of hippocampal neurons transfected with an expression vector for GFP or cotransfected with expression vectors for GFP and CaMBP4 or for GFP and CaMKIVK75E (dnCaMKIV), with DMSO or paxilline treatment. (b) Sholl analysis (left) and quantification of total dendritic length (right) in hippocampal neurons treated as indicated. Right, one-way ANOVA (P = 0.0070, F2,57 = 5.42 for DMSO group; P = 6.22 × 10–8, F2,57 = 22.51 for paxilline group) and post hoc test; unpaired t-test (P = 0.010, t38 = –2.70) for comparison between vector groups after DMSO or paxilline treatment. (c) Representative micrographs of hippocampal neurons transfected with GFP-tagged plasmids containing BK channel-specific shRNAs or non-silencing shRNAs, with DMSO or STO-609 treatment. (d) Sholl analysis (left) and quantification of total dendritic length (right) in hippocampal neurons treated as indicated. Right, unpaired t-test (P = 0.00011, t38 = 4.32 for non-silencing shRNAs group; P = 0.0015, t38 = –3.42 for BK channel-specific shRNAs group; P = 1.17 × 10–9, t38 = 7.99 for comparison between DMSO groups after non-silencing shRNAs or BK channel-specific shRNAs transfection). (e) Representative micrographs of hippocampal neurons transfected with GFP-tagged plasmids containing gene-specific shRNAs or non-silencing shRNAs, with DMSO or paxilline treatment. (f) Sholl analysis (left) and quantification of total dendritic length (right) in hippocampal neurons treated as indicated. Right, one-way ANOVA (P = 6.80 × 10–8, F7,152 = 7.64) and post hoc test. For the data in b,d,f, n = 20 cells from 4 independent cultures from at least 4 litters for each group. Fisher's least significant difference test was used for post hoc test in one-way ANOVA. Error bars represent the mean ± s.e.m.. Scale bar = 30 μm. *P < 0.05, **P < 0.01. N.S., non-silencing shRNAs.

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