Coupling of Slack and NaV1.6 sensitizes Slack to quinidine blockade and guides anti-seizure strategy development
- State Key Laboratory of Natural and Biomimetic Drugs, Department of Molecular and Cellular Pharmacology, School of Pharmaceutical Sciences, Peking University Health Science Center, China
- NHC and CAMS Key Laboratory of Medical Neurobiology, MOE Frontier Science Center for Brain Research and Brain-Machine Integration, School of Brain Science and Brain Medicine, Zhejiang University, China
- Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy, Purdue University, United States
- Department of Biophysics, Kidney Disease Center of the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China
- IDG/McGovern Institute for Brain Research, Peking University, China
Figures
Figure 1 with 3 supplements
NaV1.6 specifically sensitizes Slack to quinidine blockade.
(A) The voltage protocol and current traces from control (non-transfected) HEK293 cells. The arrows on the voltage protocol indicate the onset of inward sodium currents through NaV channels and …
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Figure 1—source data 1
Numerical data for Figure 1 and Figure 1—figure supplements 1–3.
- https://cdn.elifesciences.org/articles/87559/elife-87559-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
The sensitivity of Slack to quinidine blockade upon expression of Slack alone and co-expression of Slack with sodium-permeable channels.
(A) The inhibitory effects of 30 μΜ quinidine on Slack upon expression of Slack alone (n = 9) and co-expression of Slack with NaV1.1 (n = 6), NaV1.2 (n = 9), NaV1.3 (n = 11), NaV1.6 (n = 7), GluA1 …
Figure 1—figure supplement 2
The sensitivity of Slick to quinidine blockade upon expression of Slick alone or co-expression of Slick with NaV1.6.
(A) Example current traces from HEK293 cells expressing Slick alone and co-expressing Slick with NaV1.6. The left traces show the family of control currents; the right traces show the delayed …
Figure 1—figure supplement 3
The amplitudes and sensitivity to quinidine of sodium-activated potassium currents in primary cortical neurons.
(A) The amplitudes of IKNa in WT (n = 12) and NaV1.6-KO (n = 10) neurons. ns, p>0.05, unpaired two-tailed Student’s t-test. (B) The correlation between the amplitudes of IKNa in WT neurons before …
Figure 2 with 4 supplements
Blocking transient sodium influx through NaV1.6 reduces NaV1.6-mediated sensitization of Slack to quinidine blockade.
(A) Example current traces from HEK293 cells expressing Slack alone (top) and co-expressing Slack with NaV1.6 (bottom), with 100 nM tetrodotoxin (TTX) in the bath solution. The left traces show the …
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Figure 2—source data 1
Numerical data for Figure 2 and Figure 2—figure supplements 1–4.
- https://cdn.elifesciences.org/articles/87559/elife-87559-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Effects of 100 nM tetrodotoxin (TTX) on NaV1.6 and Slack currents.
(A) Example peak currents (Ipeak) at 0 mV of NaV1.6 before (black) and after (red) application of 100 nM TTX in the bath solution. (B) Example ramp sodium current (ramp INa) traces evoked during a …
Figure 2—figure supplement 2
Effects of depolarized prepulse potentials and riluzole on channels.
(A) Inactivation relationships of transient (INaT) and persistent sodium currents (INaP) of NaV1.6 in HEK293 cells. The currents were elicited by a 600 ms step test pulse to 0 mV from a 100 ms …
Figure 2—figure supplement 3
The sensitivity of NaV channel subtypes to quinidine blockade.
(A–E) Example current traces of NaV1.1 (A), NaV1.2 (B), NaV1.3 (C), NaV1.5 (D), and NaV1.6 (E) co-expressed with Slack in HEK293 cells. (F) The concentration–response curves for blocking of NaV by …
Figure 2—figure supplement 4
The dynamic properties of Slack and NaV1.6 channels.
(A) Average time constants calculated from single exponential decay fits of activation of Slack currents upon expression of Slack alone and co-expression of Slack with NaV1.6 (n = 10). (B) I–V …
Figure 3 with 1 supplement
Slack physically interacts with NaV1.6.
(A) Immunofluorescence of Slack, NaV1.2, NaV1.6 (green), and AnkG (red) in neocortex layer 5 (left) and hippocampal CA1 pyramidal cell layer (right). Confocal microscopy images were obtained from …
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Figure 3—source data 1
Numerical data for Figure 3.
- https://cdn.elifesciences.org/articles/87559/elife-87559-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
Glutathione S-transferase (GST) pull-down assay of Slack with the N- and C-termini of NaV1.6.
The GST-fused NaV1.6’s termini were separately expressed in BL21(DE3) and captured by GSH beads. The GST-fused proteins were subsequently incubated with cells lysates of HEK293T cells expressing …
Figure 4 with 1 supplement
NaV1.6’s N- and C-termini interacting with Slack is a prerequisite for NaV1.6-mediated sensitization of Slack to quinidine blockade.
(A) The sensitivity of Slack to quinidine blockade upon expression of Slack alone (n = 3) and co-expression of Slack with NaV1.6 (n = 3) from excised inside-out patches. The pipette solution …
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Figure 4—source data 1
Numerical data for Figure 4 and Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/87559/elife-87559-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
Comparison of the activation and amplitudes of NaV channel subtypes currents upon co-expressed with Slack.
(A) The activation time constants of peak sodium currents in HEK293 cells co-expressing NaV1.2 (n = 6), NaV1.5 (n = 5), and NaV1.6 (n = 5) with Slack, respectively. ns, p>0.05, one-way ANOVA …
Figure 5
Slack’s C-terminus is required for NaV1.6-mediated sensitization of Slack to quinidine blockade.
(A) Domain architecture of the human Slack channel subunit. Slack’s N-terminus (Slack-N, residues 1–116) and C-terminus (Slack-C, residues 345–1235) are shown in the blue boxes. (B) The …
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Figure 5—source data 1
Numerical data for Figure 5.
- https://cdn.elifesciences.org/articles/87559/elife-87559-fig5-data1-v1.xlsx
Figure 6 with 1 supplement
NaV1.6 sensitizes epilepsy-related Slack mutant variants to quinidine blockade.
(A) Co-IP of 3×Flag-tagged Slack or its mutations (Slack-3×Flag) with 3×HA-tagged NaV1.6 (NaV1.6–3×HA) in HEK293T cell lysates. The tags were all fused to the C-terminal region of wild-type or …
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Figure 6—source data 1
Numerical data for Figure 6 and Figure 6—figure supplement 1.
- https://cdn.elifesciences.org/articles/87559/elife-87559-fig6-data1-v1.xlsx
Figure 6—figure supplement 1
The Na+-mediated currents coupling of Slack mutant variants and NaV1.6 upon co-expression in HEK293 cells.
(A) Comparison of NaV1.6 sodium current amplitudes upon expression of NaV1.6 alone (n = 11) and co-expression of NaV1.6 with epilepsy-related Slack mutant variants (SlackK629N [n = 17], SlackR950Q …
Figure 7 with 1 supplement
Viral expression of Slack’s C-terminus prevents SlackG269S-induced seizures.
(A, B) The current densities of Slack mutant variants (SlackG288S [A] and SlackR398Q [B]) upon co-expression with NaV1.5/6NC in HEK293T cells were reduced by additional expression of Slack’s …
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Figure 7—source data 1
Numerical data for Figure 7 and Figure 7—figure supplement 1.
- https://cdn.elifesciences.org/articles/87559/elife-87559-fig7-data1-v1.xlsx
Figure 7—figure supplement 1
Heterozygous knockout of NaV1.6 decreases the amplitude of slow afterhyperpolarization (AHP) in hippocampal CA1 pyramidal neurons.
(A) Action potentials elicited at 300 pA followed by a slow AHP in wild-type (WT) neurons (n = 14, black) and heterozygous NaV1.6 knockout (Scn8a+/-) neurons (n = 13, red). The amplitude of slow AHP …
Figure 8
Model for protection against seizures by quinidine blockade or disruption of the Slack-NaV1.6 interaction.
The axon initial segments (AIS)-localized Slack-NaV1.6 complex provides a plausible explanation for NaV1.6-mediated sensitization of Slack to quinidine blockade. This sensitization requires physical …
Author response image 1
The activation time constants of peak sodium currents in HEK293 cells co-expressing NaV1.
2 (n = 6), NaV1.5 (n = 5), and NaV1.6 (n = 5) with Slack, respectively. ns, p > 0.05, one-way ANOVA followed by Bonferroni’s post hoc test.
Author response image 2
Comparison of peak sodium current amplitudes of NaV1.
5 (n = 9), NaV1.5/6NC (n = 13), NaV1.5/6N (n = 10), and NaV1.6 (n = 8) upon co-expressed with Slack in HEK293 cells. ns, p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; one-way …
Author response image 3
The correlation between the inhibitory effect of quinidine and the amplitudes of baseline IKNa in WT neurons (data from manuscript Figure 1K).
r = 0.1555, p=0.6294, Pearson correlation analysis.
Author response image 4
The amplitudes of IKNa in WT and NaV1.
6-KO neurons (data from manuscript Figure 1K). ns, p > 0.05, unpaired two-tailed Student’s t test.
Author response image 5
The amplitudes of NaV1.
6 sodium currents upon co-expression of NaV1.6 with epilepsy-related Slack mutant variants (SlackK629N, SlackR950Q, and SlackK985N). ns, p>0.05, oneway ANOVA followed by Bonferroni’s post hoc test.
Author response image 6
The current amplitudes of SlackR950Q before and after bath-application of 100 nM TTX upon co-expression with NaV1.
6 in HEK293 cells (n = 5). ***p < 0.001, Two-way repeated measures ANOVA followed by Bonferroni’s post hoc test.
Additional files
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Supplementary file 1
The IC50 values for quinidine sensitivity and kinetic characteristics of Slack and NaV1.x channels.
(a) The sensitivity of Slack to quinidine blockade upon expression of Slack alone and co-expression of Slack with NaV1.x. (b) The sensitivity of NaV channel subtypes to quinidine blockade upon expression of NaV1.x alone and co-expression of NaV1.x with Slack. (c) Biophysical characteristics of NaV1.6 expressed alone and NaV1.6 upon co-expression with Slack. (d) The sensitivity of Slack mutant variants to quinidine blockade upon expression of Slack mutant variants alone and co-expression of Slack mutant variants with NaV1.6.
- https://cdn.elifesciences.org/articles/87559/elife-87559-supp1-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/87559/elife-87559-mdarchecklist1-v1.docx
