Coupling of Slack and Na_V1.6 sensitizes Slack to quinidine blockade and guides anti-seizure strategy development

Tian Yuan
Yifan Wang
Yuchen Jin
Shuai Xu
Heng Zhang
Qian Chen
Na Li
Xinyue Ma
Huifang Song
Chao Peng
Hui Yang
Ze Geng
Jie Dong
Guifang Duan
Qi Sun
Yang Yang
Fan Yang
Zhuo Huang author has email address

State Key Laboratory of Natural and Biomimetic Drugs, Department of Molecular and Cellular Pharmacology, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing, 100191, 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, Hangzhou, Zhejiang, 310058, china
Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy, Purdue University, West Lafayette, IN 47907, USA
Department of Biophysics, Kidney Disease Center of the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, 310058, China
IDG/McGovern Institute for Brain Research, Peking University, Beijing, 100871, China

https://doi.org/10.7554/eLife.87559.2

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Figures and data

Na_V1.6 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 Na_V channels and delayed outward potassium currents through Slack channels. The currents were evoked by applying 600-ms step pulses to voltages varying from -120 mV to +100 mV in 10 mV increments, with a holding potential of -90 mV and a stimulus frequency of 0.20 Hz. (B) Example current traces from HEK293 cells expressing Slack alone. The left traces show the family of control currents; the right traces show Slack currents remaining after application of 30 μΜ quinidine in the bath solution. (C-E) Example current traces from HEK293 cells co-expressing Slack with Na_V1.2 (C), Na_V1.6 (D), or Na_V1.5 (E) channels before and after application of 30 μΜ quinidine. (F) The concentration-response curves for blocking of Slack by quinidine at +100 mV upon expression of Slack alone (n = 6) and co-expression of Slack with Na_V1.1 (n = 7), Na_V1.2 (n = 10), Na_V1.3 (n = 13), Na_V1.5 (n = 9), or Na_V1.6 (n = 19). Please refer to Supplementary Table 1 for IC₅₀ values. (G-H) Delayed outward currents in primary cortical neurons from homozygous Na_V1.6 knockout C3HeB/FeJ mice (Na_V1.6-KO) (H) and the wild-type littermate controls (WT) (G). Current traces were elicited by 600-ms step pulses to voltages varying from -120 mV to +100 mV in 20 mV increments, with a holding potential of -70 mV, and recorded with different bath solutions in the following order: Na⁺-based bath solution (I_Control), replacement of external Na⁺ with Li⁺ in equivalent concentration (I_Li), washout of quinidine by Na⁺-based bath solution (I_Wash), Na⁺-based bath solution with 3 μM quinidine (I_Quid), Li⁺-based bath solution with 3 μM quinidine (I_Li+Quid). The removal and subsequent replacement of extracellular Na⁺ revealed the I_KNa in neurons. (I-J), The sensitivity of native sodium-activated potassium currents (I_KNa) to 3 μM quinidine blockade in WT (I) and Na_V1.6-KO (J) neurons. I_KNa before application of quinidine was obtained from the subtraction of I_Control and I_Li. Maintained I_KNa after application of 3 μM quinidine was obtained from the subtraction of I_Quid and I_Li+Quid. (K) Summarized amplitudes of I_KNa before and after application of 3 μM quinidine in the bath solution in WT (black, n = 12) and Na_V1.6-KO (red, n = 10) primary cortical neurons. ****p < 0.0001, Two-way repeated measures ANOVA followed by Bonferroni’s post hoc test.

Blocking transient sodium influx through Na_V1.6 reduces Na_V1.6-mediated sensitization of Slack to quinidine blockade.
(A) Example current traces from HEK293 cells expressing Slack alone (top) and co-expressing Slack with Na_V1.6 (bottom), with 100nM TTX in the bath solution. The left traces show the family of control currents; the right traces show Slack currents remaining after application of quinidine. (B) The concentration-response curves for blocking of Slack by quinidine at +100 mV upon expression of Slack alone (n = 3) and co-expression of Slack with Na_V1.6 (n = 7), with 100 nM TTX in the bath solution. (C) Top, example current traces recorded from a HEK293 cell co-expressing Slack with Na_V1.6 and evoked from a 100-ms prepulse (pre) of -90 mV. Bottom, example current traces recorded from the same cell but evoked from a 100-ms prepulse of -40 mV, before and after application of quinidine. (D) I-V curves of Slack upon co-expression with Na_V1.6. The currents were evoked from a prepulse of -90 mV (black) or -40 mV (blue). (E) The concentration-response curves for blocking of Slack by quinidine at +100 mV with a prepulse of -90 mV (black, n = 19) or -40 mV (blue, n = 5). (F) Top, example current traces recorded from a HEK293 cell co-expressing Slack and Na_V1.6 without riluzole in the bath solution. Bottom, example current traces recorded from the same cell with 20 µM riluzole in the bath solution, before and after application of quinidine. (G) I-V curves of Slack upon co-expression with Na_V1.6 before (black) and after (red) application of 20 µM riluzole into bath solution.The concentration-response curves for blocking of Slack by quinidine upon co-expression of Slack with Na_V1.6, without (n = 19) or with (n = 6) 20 µM riluzole in the bath solution. (I-J) The sensitivity of Na_V channel subtypes to quinidine blockade upon expression of Na_V alone in HEK293 cells. Example current traces (I) were evoked by a 50-ms step depolarization to 0 mV from a holding potential of -90 mV. The Concentration-response curves for blocking of Na_V channel subtypes by quinidine (J) were shown on the right panel (n = 5 for Na_V1.1, n = 3 for Na_V1.2, n = 6 for Na_V1.3, n = 6 for Na_V1.5, and n = 4 for Na_V1.6).

Slack physically interacts with Na_V1.6 in vitro and in vivo.
(A) Immunofluorescence of Slack, Na_V1.2, Na_V1.6 (green), and AnkG (red) in hippocampus CA1 (left) and neocortex (right). Confocal microscopy images were obtained from Coronal brain slices of C57BL/6 mice. The panels from top to bottom show the double staining of Slack with AnkG, Na_V1.2 with AnkG, and Na_V1.6 with AnkG, respectively. (B) Coimmunoprecipitation (Co-IP) of Slack and Na_V1.6 in cell lysates from HEK293T cells co-transfected with Slack and Na_V1.6. (C) Co-IP of Slack and Na_V1.6 in mouse brain tissue lysates. Input volume corresponds to 10% of the total lysates for Co-IP. (D) A schematic diagram showing the fluorescence-labeled Slack and Na_V1.6. mTFP1 and mVenus were fused to the C-terminal region of Slack (Slack-mTFP1) and Na_V1.6 (Na_V1.6-mVenus), respectively. (E) FRET imaging of Slack-mTFP1 and Na_V1.6-mVenus co-expressed in HEK293 cells. The emission spectra measured from the edge of cell (dotted arrows in red) are used for FRET efficiency calculation. (F) The apparent FRET efficiency measured from cells co-expressing the fluorophore-tagged ion channels or co-expressing the fluorophores. **** p < 0.0001, Mann-Whitney test. (G-H) The FRET efficiency measured from cells co-expressing the fluorophore-tagged ion channels (G), or from cells co-expressing fluorophores (H). The efficiency value was plotted as a function of the fluorescence intensity ratio between mTFP1 and mVenus (Fc/Fy). Each symbol represents a single cell. The solid curve represents the FRET model that yields the best fit; dotted curves represent models with 5% higher or lower FRET efficiencies.

Na_V1.6’s N- and C-termini interacting with Slack is a prerequisite for Na_V1.6-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 Na_V1.6 (n = 3) from excised inside-out patches. The pipette solution contained (in mM) 130 KCl, 1 EDTA, 10 HEPES and 2 MgCl₂ (pH 7.3); the bath solution contained (in mM) 140 NaCl, 1 EDTA, 10 HEPES and 2 MgCl₂ (pH 7.4). The membrane voltage was held at 0 mV and stepped to voltages varying from −100 mV to 0 mV in 10 mV increments. Example current traces were shown on the left panel. The concentration-response curves for blocking of Slack by quinidine were shown on the right panel. (B) Domain architecture of the human Na_V channel pore-forming α subunit. Calculated IC₅₀ values at +100 mV of quinidine on Slack upon co-expression with indicated cytoplasmic fragments from Na_V channels. For Na_V1.6, cytoplasmic fragments used include N-terminus (Na_V1.6-N, residues 1-132), inter domain linkers (Domain I-II linker, residues 409-753; Domain II-III linker, residues 977-1199; Domain III-IV linker, residues 1461-1523), and C-terminus (Na_V1.6-C, residues 1766-1980). For Na_V1.5, cytoplasmic fragments used include N-terminus (Na_V1.5-N, residues 1-131) and C-terminus (Na_V1.5-C, residues 1772-2016). (D) Co-IP of Slack and terminal domains of Na_V1.6 in cell lysates from HEK293T cells co-expressing 3×Flag-tagged Slack (Slack-3×Flag) and 3×HA-tagged termini of Na_V1.6 (3×HA-Na_V1.6-N or 3×HA-Na_V1.6-C). The 3×Flag tag was fused to the C-terminal region of Slack and the 3×HA tag was fused to the N-terminal region of Na_V1.6’s fragments. (E) The sensitivity of Slack to quinidine blockade upon co-expression of Slack with GFP (n = 12) or N- and C-termini of Na_V1.6 (n = 11), from whole-cell recordings. (F) The sensitivity of Slack to quinidine blockade upon co-expression of Slack with N- and C-termini of Na_V1.6, from excised inside-out recordings (n = 10, using the same protocols as in Fig. 4A). Example current traces before and after application of quinidine were shown on the left panel. The concentration-response curves were shown on the right panel. (G) A schematic diagram of the Na_V1.5-1.6 chimeric channels (Na_V1.5/6^NC and Na_V1.5/6^N) used in this study. (H) Example current traces recorded from HEK293 cells co-expressing Slack and Na_V1.5-1.6 chimeras before and after application of the indicated concentration of quinidine. (I) The concentration-response curves for blocking of Slack by quinidine upon co-expression of Slack with Na_V1.5 (n = 9), Na_V1.6 (n = 19), Na_V1.5/6^NC (n = 9), or Na_V1.5/6^N (n = 9).

Slack’s C-terminus is required for Na_V1.6-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) were shown in the blue boxes. (B) The concentration-response curves for blocking of Slack by quinidine upon additional expression of Slack’s N- or C-terminus in HEK293T cells co-expressing Slack and Na_V1.5/6^NC. (C) The concentration-response curves for blocking Slack by quinidine upon additional expression of Slack’s C-terminus in HEK293 cells co-expressing Slack and Na_V1.6. (C) Co-IP of Myc-tagged Slack’s C-terminus (Slack-C-Myc) with 3×HA-tagged Na_V1.6’s termini (3×HA-Na_V1.6-N or 3×HA-Na_V1.6-C) in HEK293T cell lysates. The 3×HA tag was fused to the N-terminal region of Na_V1.6’s fragments, and the Myc tag was fused to the C-terminal region of Slack’s fragment.

Na_V1.6 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 Na_V1.6 (Na_V1.6-3×HA) in HEK293T cell lysates. The tags were all fused to the C-terminal region of wild-type or mutant ion channels. (B-D) The sensitivity of Slack mutant variants (Slack^K629N [B], Slack^R950Q [C], and Slack^K985N [D]) to quinidine blockade upon expression of Slack mutant variants alone and co-expression of Slack mutant variants with Na_V1.6. Left, example current traces recorded from HEK293 cells expressing Slack mutant variants alone and co-expressing Slack mutant variants with Na_V1.6, before and after application of the indicated concentrations of quinidine. Right, the concentration-response curves for blocking of Slack mutant variants by quinidine upon expression of Slack mutant variants alone (n = 8 for Slack^K629N, n = 7 for Slack^R950Q, and n = 5 for Slack^K985N) and co-expression of Slack mutant variants with Na_V1.6 (n = 8 for Slack^K629N upon co-expression with Na_V1.6, n = 5 for Slack^R950Q upon co-expression with Na_V1.6, and n = 7 for Slack^K985N upon co-expression with Na_V1.6). Please refer to Supplementary Table 4 for IC₅₀ values.

Viral expression of Slack’s C-terminus prevents Slack^G269S-induced seizures.
(A-B) The current densities of Slack mutant variants (Slack^G288S [A] and Slack^R398Q [B]) upon co-expression with Na_V1.5/6^NC in HEK293T cells were reduced by additional expression of Slack’s C-terminus. Left, example current traces from HEK293T cells co-expressing Slack mutant variants and Na_V1.5/6^NC or co-expressing Slack mutant variants, Na_V1.5/6^NC, and Slack’s C-terminus. Right, summarized current densities at +100 mV. * p < 0.05, ** p < 0.01, *** p < 0.001; one-way ANOVA followed by Bonferroni’s post hoc test. (C) Architecture for expression cassettes of AAVs. (D) Top, Immunofluorescence of HA-tagged Slack^G269S (green), 3×Flag-tagged Slack’s C-terminus (red), and DAPI (blue) at 5 weeks after viral injection of Slack^G269S with Slack’s C-terminus into CA1 of mice. Bottom, study design and timeline for the stereotactic injection model. (E) Time-course of KA-induced seizure stage changes at 10-min intervals based on a modified Racine, Pinal, and Rovner scale (please refer to Methods for further details). The number of mice used: “GFP” control group (n = 11), “Slack^G269S+GFP” group (n = 12), “Slack^G269S+Slack-C” group (n = 12). “GFP” vs. “Slack^G269S+GFP”: F_(1,21) = 10.48, p = 0.0040, * p < 0.05, ** p < 0.01; “Slack^G269S+GFP” vs. “Slack^G269S+Slack-C”: F_(1,22) = 10.30, p = 0.0040, ^# p < 0.05, ^## p < 0.01. “GFP” vs. “Slack^G269S+Slack-C”: F_(1,21) = 0.09574, p = 0.7600. Repeated two-way ANOVA followed by Bonferroni’s post hoc test. (F) Total seizure score per mouse over the 2 h after KA injection of these three groups. * p < 0.05, ** p < 0.01; one-way ANOVA followed by Bonferroni’s post hoc test. (G) The percentage of mice with stage VI∼IX seizures over the 2 h after KA injection in each group. * p < 0.05; Fisher’s exact test.

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