Protein arginine methylation facilitates KCNQ channel-PIP2 interaction leading to seizure suppression
- Sungkyunkwan University School of Medicine, Korea
- Seoul National University College of Medicine, Korea
- Seoul National University Medical Research Center, Korea
- Korea Advanced Institute of Science and Technology, Korea
- Korea University, Korea
- College of Medicine, Kangwon National University, Korea
- Institute for Basic Science, Korea
- Seoul National University Hospital, Korea
- Advanced Neural Technologies, Republic of Korea
Figures
Figure 1
Spontaneous seizures and increased locomotor activities in Prmt1+/- mice.
(a) reduced expression of Prmt1 in Prmt1+/- mice, compared to WT control mice. (b) Representative traces of seizure activities: short (upper) or long (middle and lower) duration of epileptiform …
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Figure 1—source data 1
Source data for Figure 1.
- https://doi.org/10.7554/eLife.17159.004
Figure 2
Comparison of neuronal excitability in WT and Prmt1+/- mice.
(a–b) a and b panels show representative trace in the whole-cell current-clamp recording from WT (a) and Prmt1+/- (b) mature dentate GCs in response to 1-s depolarizing current injection (200 pA), …
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Figure 2—source data 1
Source data for Figure 2.
- https://doi.org/10.7554/eLife.17159.006
Figure 3 with 1 supplement
KCNQ current deficiency contributed to the persistent hyperexcitability in Prmt1+/- mice.
(a–b) Representative current traces of voltage clamp recordings from WT (a; n = 22, seven mice) and Prmt1+/- (b; n = 12, four mice) mature dentate GCs in response to the voltage protocol depicted …
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Figure 3—source data 1
Source data for Figure 3.
- https://doi.org/10.7554/eLife.17159.008
Figure 3—figure supplement 1
Changes in membrane potential in WT (+/+) and Prmt1+/- GCs (+/-) in response to 10 μM XE991.
Each connected line represents an individual neuron. Right panel gives the averaged changes. XE991 was less effective in mutants than in WT.
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Figure 3—figure supplement 1—source data 1
Source data for Figure 3—figure supplement 1.
- https://doi.org/10.7554/eLife.17159.010
Figure 4
Methylation suppression with MTA or furamidine, increases neuronal excitability via KCNQ channel.
(a–b) Representative current traces recorded from MTA- (a) or furamidine- (b) pretreated WT GCs using the voltage protocol depicted below in the absence (black) or presence (blue) of 10 μM XE991. (c)…
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Figure 4—source data 1
Source data for Figure 4.
- https://doi.org/10.7554/eLife.17159.012
Figure 5 with 5 supplements
Prmt1 binds to and methylates KCNQ2 at R333, R345, R353, and R435.
(a–c) Immunoblotting analysis showing the physical association of KCNQ2 and Prmt1. HEK293T cells were transfected with expression vectors, as indicated. Whole-cell lysates were immunoprecipitated …
Figure 5—figure supplement 1
Expression of KCNQ2 and KCNQ3 in the hippocampus of WT and Prmt1+/- mice.
https://doi.org/10.7554/eLife.17159.014
Figure 5—figure supplement 2
Identification of in vivo arginine methylation sites of KCNQ2 MS/MS spectra of the methylated peptides.
(a) 332RRNPAAGLIQSAWR345, (b) 334NPAAGLIQSAWR345, (c) 346FYATNLSR353 and (d) 435RSPSADQSLEDSPSK449. R* represents monomethylated arginine and R** represents dimethylated arginine.
Figure 5—figure supplement 3
The sequence alignment of 5 difference human KCNQ isoforms.
The arginine residues critical for methylation in KCNQ2 are marked in red. KCNQ3 and KCNQ4 have 3 conserved arginine residues while KCNQ5 and KCNQ1 have two or one conserved arginine residues, …
Figure 5—figure supplement 4
In vitro methylation assays with GST-Prmt1 or myc-Prmt5 together with GST-KCNQ2 (320-449 aa).
In vitro methylation assays with myc-Prmt5 purified from HEK293T cells and GST-Prmt1 by using GST-KCNQ2 (aa 320–449) as a substrate and histone as a positive control. Coomassie blue stainings show …
Figure 5—figure supplement 5
Expression of Prmt8 in Prmt1+/+ and Prmt1+/- brain lysates.
https://doi.org/10.7554/eLife.17159.018
Figure 6
Methylation of KCNQ2 regulates its channel activity.
(a) Representative current recordings from HEK293T cells expressing KCNQ2 with control shRNA vector (left) or a Prmt1 shRNA (Prmt1 sh) (right). Currents were elicited by voltage steps from -70 mV to …
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Figure 6—source data 1
Source data for Figure 6.
- https://doi.org/10.7554/eLife.17159.021
Figure 7 with 5 supplements
Methylation of KCNQ2 determines its PIP2 affinity.
(a) Pooled data show 10 μM neomycin-induced rundown of WT (●) and R333K (○) KCNQ2 currents. KCNQ2 currents were normalized to KCNQ2 current at t = 0. (b) Dose-response curves for neomycin measured …
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Figure 7—source data 1
Source data for Figure 7.
- https://doi.org/10.7554/eLife.17159.023
Figure 7—figure supplement 1
Surface expression of KCNQ channels was not affected by Prmt1 knockdown or 4RK mutation.
Membrane surface biotinylation assays show that the ratio of channels on the membrane to those in the total lysates was not significantly different in control cells and Prmt1 knockdown cells (left). …
Figure 7—figure supplement 2
Quantitative determination of the sensitivity of KCNQ2 channels to activation of Dr-VSP in HEK293T cells.
Tail current amplitudes were used to measure current inhibition by Dr-VSP activation and its recovery. (a–e) Time course of tail current amplitude in a cell transfected with Dr-VSP and WT KCNQ2 (a), …
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Figure 7—figure supplement 2—source data 1
Source data for Figure 7—figure supplement 2.
- https://doi.org/10.7554/eLife.17159.026
Figure 7—figure supplement 3
Comparison of XE991 sensitivity in GCs.
Changes in firing rate (a) and threshold current (b) in WT (+/+) neurons, mutants (+/-), and mutants loaded with 20μM diC8-PIP2 (+/-; PIP2) in response to 10 μM XE991. Each connected line represents …
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Figure 7—figure supplement 3—source data 1
Source data for Figure 7—figure supplement 3.
- https://doi.org/10.7554/eLife.17159.028
Figure 7—figure supplement 4
Application of exogenous PIP2 had little effect on the excitability in WT neurons.
(a) representative trace in the whole-cell current-clamp recording from WT GCs with no PIP2 (left) or 20 μM diC8-PIP2 in the patch pipette (right) in response to 1-s depolarizing current injection …
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Figure 7—figure supplement 4—source data 1
Source data for Figure 7—figure supplement 4.
- https://doi.org/10.7554/eLife.17159.030
Figure 7—figure supplement 5
The effects of SK channel block and BK channel block on neuronal excitability of control or furamidine-pretreated GCs.
Spike trains were evoked by injecting 1-s depolarizing current pulses of different intensities (100–250 pA) into the cell before and after application of apamin (a–f) or paxilline (g–l) in control …
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Figure 7—figure supplement 5—source data 1
Source data for Figure 7—figure supplement 5.
- https://doi.org/10.7554/eLife.17159.032
Figure 8
Prmt1 is involved in Oxidative stress-induced silencing of neurons.
Spike trains were evoked by injecting 1-s depolarizing current pulses of different intensities (100–250 pA) into the cell before (black) and after (red) application of H2O2. Left panels show …
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Figure 8—source data 1
Source data for Figure 8.
- https://doi.org/10.7554/eLife.17159.034
Figure 9 with 1 supplement
Activation of KCNQ2 channels by H2O2 is sensitive to methylation.
(a–g) Time course for the effect of 500 μM H2O2 on KCNQ2 currents. WT KCNQ2 (a-c), R333K (d), R345K (e), R353K (f) or R435K (g) were expressed in HEK293T cells. For experiment (b) & (c), cells were …
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Figure 9—source data 1
Source data for Figure 9.
- https://doi.org/10.7554/eLife.17159.036
Figure 9—figure supplement 1
Time course for the effect of 500 μM H2O2 on Triple-Cys mutant of KCNQ2 currents (left).
Summarized data for Triple-Cys mutant of KCNQ2 currents activation induced by H2O2 or retigabine (right). (b) Treatment of MTA induces a reduction of KCNQ2(WT) and Triple-Cys mutant of KCNQ2 …
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Figure 9—figure supplement 1—source data 1
Source data for Figure 9—figure supplement 1.
- https://doi.org/10.7554/eLife.17159.038
Tables
Table 1
Mono- and dimethylated peptides identified from KCNQ2.
| Peptide* | Modification | Charge state |
|---|---|---|
| 332RRNPAAGLIQSAWR345 | Mono-methylated R333 | +3 |
| 334NPAAGLIQSAWR345 | Mono-methylated R345 | +2 |
| 346FYATNLSR353 | Di-methylated R353 | +2 |
| 435RSPSADQSLEDSPSK449 | Di-methylated R435 | +3 |
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*Methylated amino acid residues are highlighted in bold.
