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 Ca2+ 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 Ca2+ release, nucleoplasmic Ca2+ elevation and cyclic AMP response element binding protein (CREB)-dependent transcription. More importantly, blockade of nBK channels regulates nuclear Ca2+–sensitive gene expression and promotes dendritic arborization in a nuclear Ca2+–dependent manner. These results suggest that the nBK channel functions as a molecular link between neuronal activity and nuclear Ca2+ to convey signals from synapse to nucleus and is a new modulator, operating at the NE, of synaptic activity–dependent neuronal functions.
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We thank X. Tang (Nankai University) for BK-GFP plasmid, M. Nathanson (Yale University) for PV-NLS plasmid, H. Bading (University of Heidelberg) for CaMKIVK75E and CaMBP4 plasmids, and F.-X. Liang and the Microscopy Core of New York University Medical Center for immunoelectron microscopy analysis. This work was supported by the National Natural Science Foundation of China (grants 81030022, 81329003 and U1201225 to T.-M.G., 30900581 to B.L., 81001129 to L.H.), the Program for Changjiang Scholars and Innovative Research Team (grant IRT1142), Guangdong Natural Science Foundation (grants 9351051501000003 and CXZD1018 to T.-M.G., 10451051501004726 to L.H.), Guangzhou Science and Technology Project (grant 7411802013939), Medical Scientific Research Foundation of Guangdong Province (B2010170 to L.H.) and the US National Heart, Lung, and Blood Institute (grant R01-HL102758 to A.L.M.).
The authors declare no competing financial interests.
Integrated supplementary information
The distribution of BK channel β4 subunits (red) was examined by confocal immunofluorescence in cultured hippocampal neurons. Intracellular ring-like labeling around the nucleus was observed and colocalized with lamin B (green). Scale bar represents 20 μm.
Supplementary Figure 2 The effects of BK channel shRNAs on nBK channel expression and nuclear Ca2+ signal.
(a) BK channel expression after knockdown by two pairs of lentiviral shRNAs. (b) Paxilline-induced changes in Fluo-4 AM fluorescence intensity in the isolated nuclei after knockdown of BK channel (n = 25 nuclei for each group, unpaired t-test, P = 1.83 × 10−7, t48 = 6.09). (c) Paxilline-induced changes in Fluo-4 dextran fluorescence intensity in the isolated nuclei after knockdown of BK channel (n = 25 nuclei for each group, unpaired t-test, P = 2.67 × 10−7, t48 = 5.98). The bottom and top of the box represent the first and third quartiles, and the band inside the box is the median. The whisker represents the minimum and maximum of all of the data. **P < 0.01. The full-length blots for a are presented in Supplementary Figure 7.
(a) RyRs mRNA level after knockdown by shRNAs. GAPDH was used as control. One-way ANOVA (N = 3 for each group, P = 9.47 × 10−6, F3,8 = 57.27) and post hoc test. (b) RyRs protein level after knockdown by shRNAs. 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. The full-length blots for b are presented in Supplementary Figure 7.
(a) Outward potassium channel currents were recorded in whole-cell patch clamp to show that paxilline (10 μM) had no additive effect on pmBK channel blockade by IbTx (100 μM). The recording solution contains 1mM 4-AP, 1μM TTX, and 5μM glybenclamide. (b) The effects of IbTx (100 μM) and IbTx (100 μM)+paxilline (10 μM) on action potentials. IbTx broadened the action potential by blocking BK channels. Paxilline did not show additive effects. (c) Pre-incubation (10 min) of IbTx did not affect BK channel expression on plasma membrane. Membrane proteins were isolated as described in Methods section. Na/K ATPase was used as loading control. The full-length blots for c are presented in Supplementary Figure 7.
mRNA level of Fos, Npas4, Atf3, Btg2, Bcl6 and Ifi202b after knockdown by shRNAs as indicated. One-way ANOVA (N = 3 independent cultures from at least 3 litters for each group; P = 1.77 × 10−5, F2,6 = 112.05 for Fos; P = 1.70 × 10−6, F2,6 = 248.60 for Npas4; P = 2.47 × 10−5, F2,6 = 99.99 for Atf3; P = 1.09 × 10−5, F2,6 = 132.38 for Btg2; P = 2.84 × 10−5, F2,6 = 95.38 for Bcl6; P = 1.09 × 10−5, F2,6 = 132.29 for Ifi202b) 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..
(a) Representative micrographs of hippocampal neurons transfected with GFP-tagged plasmids containing gene-specific shRNAs or non-silencing shRNAs with the treatment of DMSO. (b) Sholl analysis (left) and quantification of total dendritic length (right) in hippocampal neurons treated as indicated. Right, one-way ANOVA (P = 0.042, F6,133 = 2.26) and post hoc test. 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 < 0.01. N.S., non-silencing shRNAs.
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