PRRT2 as an auxiliary regulator of Nav channel slow inactivation
- Center for Excellence in Brain Science and Intelligence Technology (Institute of Neuroscience), Chinese Academy of Sciences, China
- University of Chinese Academy of Sciences, China
- School of Life Science and Technology, ShanghaiTech University, China
- Department of Neurology, Zhongshan Hospital, Fudan University, China
- Department of Medical Genetics and Center for Rare Diseases, Second Affiliated Hospital, Zhejiang University School of Medicine and Zhejiang Key Laboratory of Rare Diseases for Precision Medicine and Clinical Translation, China
- Shanghai Center for Brain Science and Brain-Inspired Technology, China
Figures
Figure 1 with 1 supplement
PRRT2 regulates slow inactivation of Nav1.2 channels.
(A, B) Representative immunoblots showing PRRT2 protein expression (A) and schematic of whole-cell recording (B) in Nav1.2-stably expressing HEK293T cells transfected with constructs encoding either EGFP or mouse PRRT2 (mPRRT2). MW, molecular weight. (C) Fast-inactivation decay time constants of sodium currents evoked by 20 ms depolarizing steps to the indicated command voltages (EGFP: n=13 cells, mPRRT2: n=13 cells). Insert: representative sodium current traces elicited by a brief depolarization (left) and voltage-clamp protocol used to assess fast inactivation (right). The arrow indicates the decay of the sodium current attributable to fast inactivation. (D) Recovery from fast inactivation of Nav1.2 channels (EGFP: n=10 cells, mPRRT2: n=7 cells). Insert: Protocol for examining fast inactivation recovery. (E) Schematic diagram of entry into and recovery from slow inactivation of voltage-gated sodium channels. Slow inactivation modulates the availability of the sodium channels. (F) Entry into slow-inactivated state of Nav1.2 channels induced by progressively longer depolarization (EGFP: n=6 cells, mPRRT2: n=6 cells). (G) The effects of mPRRT2 on the recovery of Nav1.2 channels from slow-inactivated state induced by 5 s depolarization (EGFP: n=6 cells, mPRRT2: n=6 cells). Data are presented as mean ± s.e.m. Two-tailed, unpaired Student’s t-test was used in (C), and two-way ANOVAs were used in (D), (F), and (G). ****p<0.0001. n.s., not significant. Source data are provided in Figure 1—source data 1–3.
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Figure 1—source data 1
Original western blot images for panel A.
- https://cdn.elifesciences.org/articles/109327/elife-109327-fig1-data1-v1.zip
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Figure 1—source data 2
PDF file containing original western blot images with the relevant bands labeled for panel A.
- https://cdn.elifesciences.org/articles/109327/elife-109327-fig1-data2-v1.zip
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Figure 1—source data 3
Raw data used for the quantifications shown in panels C, D, F, and G.
- https://cdn.elifesciences.org/articles/109327/elife-109327-fig1-data3-v1.xlsx
Figure 1—figure supplement 1
PRRT2 promotes steady-state slow inactivation and use-dependent inactivation.
(A) Protocol of steady-state slow inactivation test (left) and representative traces of the sodium currents evoked by test pulses (right). The sodium currents gradually decreased as the voltage in conditioning steps increased. Scale bar, 0.5 nA, 2 ms. (B) The effects of PRRT2 on voltage-dependent steady-state slow inactivation of Nav1.2 channels (EGFP: n=11 cells, mPRRT2: n=13 cells). mPRRT2, mouse PRRT2. (C) PRRT2 modulates use-dependent inactivation of Nav1.2 channels during pulse trains (EGFP: n=6 cells, mPRRT2: n=6 cells). Data are presented as mean ± s.e.m. Two-way ANOVAs were used in (B, C) to determine the statistical significance of main effects of group. ****p<0.0001. Source data are provided in Figure 1—figure supplement 1—source data 1.
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Figure 1—figure supplement 1—source data 1
Raw data used for the quantifications shown in panels B and C.
- https://cdn.elifesciences.org/articles/109327/elife-109327-fig1-figsupp1-data1-v1.xlsx
Figure 2 with 1 supplement
Functional auxiliary factors in the regulation of Nav channel slow inactivation.
(A) Diagram of Nav channel with potent auxiliary factors, encompassing SCN1B, FHF2A (also known as FGF13a), and PRRT2. (B) The effects of auxiliary factors on the entry of Nav1.2 channels into slow inactivation (EGFP: n=12 cells, mPRRT2: n=14 cells, SCN1B: n=9 cells, FGF13a: n=12 cells). (C) The effects of auxiliary factors on the recovery of Nav1.2 channels from slow-inactivated state induced by 5 s depolarization (EGFP: n=12 cells, mPRRT2: n=12 cells, SCN1B: n=12 cells, FGF13a: n=12 cells). (D) Schematic representation of the putative topology of mouse PRRT2 and three truncations. Numbers indicate the positions of the amino acid. REL, re-entrant loop. TM, transmembrane domain. N, amino terminus. C, carboxyl terminus. aa, amino acid. (E) Effects of PRRT2 truncations on the entry of Nav1.2 channels into slow inactivation (EGFP: n=5 cells, mPRRT2(1–266): n=14 cells, mPRRT2(222–346): n=12 cells, mPRRT2(256–346): n=12 cells). (F) Effects of PRRT2 truncations on the recovery of Nav1.2 channels from slow-inactivated state induced by 5 s depolarization (EGFP: n=5 cells, mPRRT2(1–266): n=10 cells, mPRRT2(222–346): n=10 cells, mPRRT2(256–346): n=12 cells). Data are presented as mean ± s.e.m. The main effect of group was assessed using two-way ANOVAs (B, C, E, F). **p<0.01, ****p<0.0001, n.s., not significant. Source data are provided in Figure 2—source data 1.
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Figure 2—source data 1
Raw data used for the quantifications shown in panels B, C, E, and F.
- https://cdn.elifesciences.org/articles/109327/elife-109327-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Western blot verification of mPRRT2(1–266) expression.
(A) Representative immunoblot showing HA-tagged mPRRT2 (1–266) expression in Nav1.2-stably expressing HEK293T cells. GAPDH served as a loading control. (B) Quantification of mPRRT2(1–266) HA expression normalized to GAPDH (n=2 independent samples). Data are presented as mean ± s.e.m. Source data are provided in Figure 2—figure supplement 1—source data 1–3.
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Figure 2—figure supplement 1—source data 1
Original western blot images for panel A.
- https://cdn.elifesciences.org/articles/109327/elife-109327-fig2-figsupp1-data1-v1.zip
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Figure 2—figure supplement 1—source data 2
PDF file containing original western blot images with the relevant bands labeled for panel A.
- https://cdn.elifesciences.org/articles/109327/elife-109327-fig2-figsupp1-data2-v1.zip
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Figure 2—figure supplement 1—source data 3
Raw data used for the quantification shown in panel B.
- https://cdn.elifesciences.org/articles/109327/elife-109327-fig2-figsupp1-data3-v1.xlsx
Figure 3 with 1 supplement
Evolutionarily conserved effects of PRRT2 on Nav channel slow inactivation.
(A) Sequence alignment of PRRT2 protein in human, mouse, and zebrafish. Conserved amino acids are highlighted and the membrane-associated domains in the carboxyl terminus of PRRT2 are underlined. REL, re-entrant loop. TM, transmembrane domain. (B) Sequence identity of PRRT2 protein in human (hPRRT2), mouse (mPRRT2), and zebrafish (zfPRRT2). Sequence identity is calculated by using the protein BLAST tool from NCBI. (C) The effects of PRRT2 from different species on the entry of Nav1.2 channels into slow inactivation (EGFP: n=16 cells, mPRRT2: n=19 cells, hPRRT2: n=20 cells, zfPRRT2: n=11 cells). (D) The effects of PRRT2 from different species on the recovery of Nav1.2 channels from the slow-inactivated state induced by 5 s depolarization (EGFP: n=16 cells, mPRRT2: n=19 cells, hPRRT2: n=19 cells, zfPRRT2: n=11 cells). (E) Diagram for the chimeric construct of PRRT2 originated from human and zebrafish. (F) The effect of chimeric PRRT2 on the entry of Nav1.2 channels into slow inactivation (EGFP: n=11 cells, hPRRT2: n=6 cells, zfPRRT2: n=10 cells, Chimera: n=11 cells). (G) The effect of chimeric PRRT2 on the recovery of Nav1.2 channels from the slow-inactivated state induced by 5 s depolarization (EGFP: n=11 cells, hPRRT2: n=6 cells, zfPRRT2: n=10 cells, Chimera: n=9 cells). Data are presented as mean ± s.e.m. The main effect of group was assessed using two-way ANOVAs (C, D, F, G). **p<0.01, ****p<0.0001, n.s., not significant. Source data are provided in Figure 3—source data 1.
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Figure 3—source data 1
Raw data used for the quantifications shown in panels C, D, F, and G.
- https://cdn.elifesciences.org/articles/109327/elife-109327-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
Paralogs of PRRT2 regulate Nav channel slow inactivation.
(A) Sequence alignment of mouse TRARG1, TMEM233, and PRRT2 proteins. Conserved amino acids are highlighted and the transmembrane domains in the carboxyl terminus of these proteins are underlined. REL, re-entrant loop. TM, transmembrane domain. (B) Schematic showing the phylogenetic relationship between members of the Dispanin subfamily B (DspB). (C) The effects of members of DspB on the entry of Nav1.2 channels into slow inactivation (EGFP: n=13 cells, TRARG1: n=17 cells, TMEM233: n=13 cells, PRRT2: n=13 cells). (D) The effects of members of DspB on the recovery of Nav1.2 channels from slow-inactivated states induced by 5 s depolarization (EGFP: n=10 cells, TRARG1: n=14 cells, TMEM233: n=12 cells, PRRT2: n=12 cells). Data are presented as mean ± s.e.m. The main effect of group was assessed using two-way ANOVAs (C, D). **p<0.01, ****p<0.0001, n.s., not significant. Source data are provided in Figure 3—figure supplement 1—source data 1.
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Figure 3—figure supplement 1—source data 1
Raw data used for the quantifications shown in panels C and D.
- https://cdn.elifesciences.org/articles/109327/elife-109327-fig3-figsupp1-data1-v1.xlsx
Figure 4
PRRT2 promotes slow inactivation across multiple human Nav channel isoforms.
(A) Protocol for examining the entry of Nav channels into slow-inactivated state. (B–E) The effects of human PRRT2 (hPRRT2) on the entry of Nav1.1, Nav1.4, Nav1.5, and Nav1.6 channels into slow inactivation. In (B), analyses for Nav1.1 (EGFP: n=10 cells, hPRRT2: n=12 cells); in (C), analyses for Nav1.4 (EGFP: n=11 cells, hPRRT2: n=11 cells); in (D), analyses for Nav1.5 (EGFP: n=17 cells, hPRRT2: n=17 cells); in (E), analyses for Nav1.6 (EGFP: n=5 cells, hPRRT2: n=4 cells). (F) Protocol for examining the recovery of Nav channels from slow-inactivated state. (G–J) The effects of human PRRT2 on the recovery of Nav1.1, Nav1.4, Nav1.5, and Nav1.6 channels from slow-inactivated state induced by 5 s depolarization. In (G), analyses for Nav1.1 (EGFP: n=10 cells, hPRRT2: n=12 cells); in (H), analyses for Nav1.4 (EGFP: n=11 cells, hPRRT2: n=11 cells); in (I), analyses for Nav1.5 (EGFP: n=16 cells, hPRRT2: n=15 cells); in (J), analyses for Nav1.6 (EGFP: n=5 cells, hPRRT2: n=4 cells). Data are presented as mean ± s.e.m. The main effect of group was assessed using two-way ANOVAs (B–E and G–J). **p<0.01, ***p<0.001, ****p<0.0001. Source data are provided in Figure 4—source data 1.
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Figure 4—source data 1
Raw data used for the quantifications shown in panels B, C, D, E, G, H, I, and J.
- https://cdn.elifesciences.org/articles/109327/elife-109327-fig4-data1-v1.xlsx
Figure 5
Association between PRRT2 and Nav channels in vitro.
(A, B) Schematic diagram of the co-immunoprecipitation assay used to detect association between human PRRT2 and Nav1.2. Flag-tagged Nav1.2 (+), HA-tagged PRRT2 (+), and empty (-) vectors were transfected in HEK293T cells as indicated. The cell lysates were immunoprecipitated with anti-Flag (A) or anti-HA (B) magnetic beads, the captured proteins were analyzed by SDS-PAGE and immunoblotting. (C) Diagram for HA-tagged truncation of human PRRT2 (PRRT2(1–268)), in which the carboxyl terminus of PRRT2 was deleted. REL, re-entrant loop. TM, transmembrane domain. (D, E) Co-immunoprecipitation assay for potential association between Nav1.2 and PRRT2(1–268). The proteins immunoprecipitated by anti-Flag (D) or anti-HA (E) magnetic beads were analyzed by immunoblotting. Note that HA-tagged PRRT2(1–268) was detected by anti-HA antibody in (D, E). (F) Diagram for HA-tagged truncation of human PRRT2 (PRRT2(250–340)), in which the amino terminus of PRRT2 was deleted. (G, H) Co-immunoprecipitation assay for potential association between Nav1.2 and PRRT2(250–340). The proteins immunoprecipitated by anti-Flag (G) or anti-HA (H) were analyzed by immunoblotting. Note that HA-tagged PRRT2(250–340) was detected by anti-HA antibody in (G, H). (I, J) Co-immunoprecipitation assay for potential association between Nav1.1 and PRRT2. The proteins captured by anti-Flag (I) or anti-HA (J) magnetic beads were analyzed by immunoblotting. In (A, B, D, E, G, H, I, J), blots shown are representative of at least three independent co-immunoprecipitation experiments with similar results. Red rectangles indicate the immunoblotting of bait and potential prey proteins in co-immunoprecipitation. IB, immunoblot. IP, immunoprecipitation. MW, molecular weight. Source data are provided in Figure 5—source data 1–16.
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Figure 5—source data 1
Original western blot images for panel A.
- https://cdn.elifesciences.org/articles/109327/elife-109327-fig5-data1-v1.zip
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Figure 5—source data 2
PDF file containing original western blot images with the relevant bands labeled for panel A.
- https://cdn.elifesciences.org/articles/109327/elife-109327-fig5-data2-v1.zip
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Figure 5—source data 3
Original western blot images for panel B.
- https://cdn.elifesciences.org/articles/109327/elife-109327-fig5-data3-v1.zip
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Figure 5—source data 4
PDF file containing original western blot images with the relevant bands labeled for panel B.
- https://cdn.elifesciences.org/articles/109327/elife-109327-fig5-data4-v1.zip
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Figure 5—source data 5
Original western blot images for panel D.
- https://cdn.elifesciences.org/articles/109327/elife-109327-fig5-data5-v1.zip
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Figure 5—source data 6
PDF file containing original western blot images with the relevant bands labeled for panel D.
- https://cdn.elifesciences.org/articles/109327/elife-109327-fig5-data6-v1.zip
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Figure 5—source data 7
Original western blot images for panel E.
- https://cdn.elifesciences.org/articles/109327/elife-109327-fig5-data7-v1.zip
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Figure 5—source data 8
PDF file containing original western blot images with the relevant bands labeled for panel E.
- https://cdn.elifesciences.org/articles/109327/elife-109327-fig5-data8-v1.zip
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Figure 5—source data 9
Original western blot images for panel G.
- https://cdn.elifesciences.org/articles/109327/elife-109327-fig5-data9-v1.zip
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Figure 5—source data 10
PDF file containing original western blot images with the relevant bands labeled for panel G.
- https://cdn.elifesciences.org/articles/109327/elife-109327-fig5-data10-v1.zip
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Figure 5—source data 11
Original western blot images for panel H.
- https://cdn.elifesciences.org/articles/109327/elife-109327-fig5-data11-v1.zip
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Figure 5—source data 12
PDF file containing original western blot images with the relevant bands labeled for panel H.
- https://cdn.elifesciences.org/articles/109327/elife-109327-fig5-data12-v1.zip
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Figure 5—source data 13
Original western blot images for panel I.
- https://cdn.elifesciences.org/articles/109327/elife-109327-fig5-data13-v1.zip
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Figure 5—source data 14
PDF file containing original western blot images with the relevant bands labeled for panel I.
- https://cdn.elifesciences.org/articles/109327/elife-109327-fig5-data14-v1.zip
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Figure 5—source data 15
Original western blot images for panel J.
- https://cdn.elifesciences.org/articles/109327/elife-109327-fig5-data15-v1.zip
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Figure 5—source data 16
PDF file containing original western blot images with the relevant bands labeled for panel J.
- https://cdn.elifesciences.org/articles/109327/elife-109327-fig5-data16-v1.zip
Figure 6 with 1 supplement
PRRT2 forms a molecular complex with Nav1.2 in the mouse brain.
(A) Schematic showing the generation of Prrt2-V5 knock-in (V5-KI) mice using Cas9 technology. V5-tag sequence was inserted precisely at the end of Prrt2 gene. PAM, protospacer-adjacent motif (NGG). ssDNA, single-stranded DNA. (B) Sanger sequencing around sgRNA targeting site in Prrt2-V5 knock-in mouse. The V5-tag, stop codon, and homologous arms were denoted. (C) Co-immunoprecipitation assay for potential interaction between Nav1.2 and PRRT2-V5 in the brain tissue. The proteins immunoprecipitated by anti-V5 nanobody were analyzed by immunoblotting. Blots shown are representative of four independent co-immunoprecipitation experiments. IB, immunoblot. IP, immunoprecipitation. MW, molecular weight. WT, wild-type. KI, knock-in. (D–F) Quantification of the density of protein bands for PRRT2 (D) and Nav1.2 (E) in lysate, and for Nav1.2 and ATP1B2 in co-immunoprecipitation experiments (F) (n=4 mice for each group). Data are presented as mean ± s.e.m. In (D–F), two-tailed, paired Student’s t-test was used for determining statistical significance. **p<0.01, ****p<0.0001, n.s., not significant. Source data are provided in Figure 6—source data 1–3.
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Figure 6—source data 1
Original western blot images for panel C.
- https://cdn.elifesciences.org/articles/109327/elife-109327-fig6-data1-v1.zip
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Figure 6—source data 2
PDF file containing original western blot images with the relevant bands labeled for panel C.
- https://cdn.elifesciences.org/articles/109327/elife-109327-fig6-data2-v1.zip
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Figure 6—source data 3
Raw data used for the quantifications shown in panels D–F.
- https://cdn.elifesciences.org/articles/109327/elife-109327-fig6-data3-v1.xlsx
Figure 6—figure supplement 1
Dystonia-like behaviors in a subset of Prrt2-V5 knock-in mice.
(A) Schematic illustration of electrical stimulation delivered to the localized area of the fourth/fifth cerebellar lobule in mice. (B) Representative postures of wild-type (WT) and Prrt2-V5 knock-in (KI) mice following cerebellar stimulation. Arrows indicate the body parts affected during the dystonic episode. (C) Incidence of dystonia-like behaviors induced by cerebellar electrical stimulation in WT, Prrt2-V5 KI, and Prrt2-mutant mice (WT: n=6 mice, Prrt2-V5 KI: n=5 mice, Prrt2-mutant: n=4 mice). Data are presented as percentage of animals that exhibited dystonia-like behaviors within 5 min after stimulation. Source data are provided in Figure 6—figure supplement 1—source data 1.
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Figure 6—figure supplement 1—source data 1
Raw data used for the quantification shown in panel C.
- https://cdn.elifesciences.org/articles/109327/elife-109327-fig6-figsupp1-data1-v1.xlsx
Figure 7 with 2 supplements
PRRT2 deficiency impairs the regulation of Nav channel slow inactivation and neuronal resilience in mice.
(A) Schematic showing the isolated axonal bleb recording in cortical neuron (left) and the representative brightfield image for the axonal bleb in brain slice (middle). The arrow indicates an axonal bleb. Scale bar, 20 μm. The representative sodium current traces recorded in axonal blebs (right). Scale bar, 0.5 nA, 5 ms. TTX, tetrodotoxin. (B) Sodium current density recorded in isolated axonal blebs from wild-type (WT) and Prrt2-mutant mice (WT: n=18 blebs from 7 mice, Prrt2-mutant: n=20 blebs from 8 mice). Sodium currents were measured with a brief depolarization to 0 mV preceded by a 200 ms hyperpolarizing prepulse to –110 mV. (C) Protocol for assessing Nav channel slow-inactivation (upper) and the representative traces of the sodium currents evoked by conditioning depolarization pulse and test pulses (bottom). Scale bar, 0.5 nA, 5 ms. (D) Fraction of available Nav channels after 5 s depolarization (WT: n=18 blebs from 7 mice, Prrt2-mutant: n=19 blebs from 8 mice). (E) Schematic illustration of the cortical electrostimulation and EEG recording in mice. Ref, reference electrode. Gnd, ground electrode. Stim, Stimulation. (F) Illustration showing the experimental schedule and the stimulation parameters. Stimulation was delivered once daily with stepwise increases in current intensity. (G) Representative traces and power of EEG signals in wild-type and Prrt2-mutant mice before and after 2 s cortical stimulation. After-discharges are indicated by the arrow and red line. Scale bar: 0.2 mV. Electrical stimulation (120 µA) was applied during 60–62 s period, where the stimulus-induced artifacts were removed prior to analysis. (H) Threshold of electrical stimulation to induce after-discharges in WT and Prrt2-mutant mice (WT: n=16 mice, Prrt2-mutant: n=8 mice). (I) Percentage of after-discharge occurrence in WT and Prrt2-mutant mice after electrical stimulation (WT: n=16, Prrt2-mutant: n=8). Data are presented as mean ± s.e.m. Two-tailed, unpaired Student’s t-test was used in (B, D) and two-tailed, unpaired Mann–Whitney test was used in (H). In (I), the main effect of group was assessed using two-way ANOVAs. ****p<0.0001, n.s., not significant. Source data are provided in Figure 7—source data 1.
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Figure 7—source data 1
Raw data used for quantifications in panels B, D, H, and I.
- https://cdn.elifesciences.org/articles/109327/elife-109327-fig7-data1-v1.xlsx
Figure 7—figure supplement 1
Protein levels of Nav1.2 are unchanged in brain tissue from Prrt2-mutant mice.
(A, B) Immunoblots (A) and quantifications (B) of Nav1.2 proteins in brain tissue of wild-type (WT) and Prrt2-mutant mice (n=3 mice). Data are presented as mean ± s.e.m. Two-tailed, paired Student’s t-test was used in (B) to determine the statistical significance. n.s., not significant. Source data are provided in Figure 7—figure supplement 1—source data 1–3.
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Figure 7—figure supplement 1—source data 1
Original western blot images for panel A.
- https://cdn.elifesciences.org/articles/109327/elife-109327-fig7-figsupp1-data1-v1.zip
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Figure 7—figure supplement 1—source data 2
PDF file containing original western blot images with the relevant bands labeled for panel A.
- https://cdn.elifesciences.org/articles/109327/elife-109327-fig7-figsupp1-data2-v1.zip
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Figure 7—figure supplement 1—source data 3
Raw data used for the quantification shown in panel B.
- https://cdn.elifesciences.org/articles/109327/elife-109327-fig7-figsupp1-data3-v1.xlsx
Figure 7—figure supplement 2
Activity-dependent adaptation is impaired in Prrt2-mutant mice.
(A) Schematic illustration of corpus callosum stimulation and compound action potential (AP) recording in acute brain slices. Insert: representative trace of a compound AP recorded from the corpus callosum. Scale bar, 0.2 mV, 1 ms. (B, C) Normalized peak amplitude of compound APs in corpus callosum during repetitive electrical stimulation (100 μA) at 1 Hz (B) and 20 Hz (C) in WT and Prrt2-mutant mice (WT: n=22 slices from 5 mice, Prrt2-mutant: n=20 slices from 4 mice). Inserts: representative traces of the 1st and 100th compound APs recorded from the corpus callosum of WT and Prrt2-mutant mice. Scale bar, 0.2 mV, 1 ms. Data are presented as mean ± s.e.m. Two-way ANOVAs were used in (B) and (C) to determine the statistical significance of the main effect of genotype. ****p<0.0001. n.s., not significant. Source data are provided in Figure 7—figure supplement 2—source data 1.
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Figure 7—figure supplement 2—source data 1
Raw data used for the quantifications shown in panels B and C.
- https://cdn.elifesciences.org/articles/109327/elife-109327-fig7-figsupp2-data1-v1.xlsx
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Homo sapiens) | Human PRRT2 | GenBank | 112476 | |
| Gene (Mus musculus) | Mouse Prrt2 | GenBank | 69017 | |
| Gene (Danio rerio) | Zebrafish Prrt2 | GenBank | 100329612 | |
| Gene (M. musculus) | Tmem233 | OriGene Technologies | Cat#:MR214829 | Also known as DSPB2 |
| Gene (M. musculus) | Trarg1 | OriGene Technologies | Cat#:MR218173 | Also known as DSPB1, Tusc5 |
| Gene (M. musculus) | Scn1b | OriGene Technologies | Cat#:MR202467 | |
| Gene (M. musculus) | Fgf13 | OriGene Technologies | Cat#:MR 221854 | Also known as Fhf2 |
| Genetic reagent (M. musculus, male and female) | Prrt2-mutant | Tan et al., 2018; PMID:29056747 | Zhi-Qi Xiong’s Laboratory | |
| Genetic reagent (M. musculus, male and female) | Prrt2-V5 knock-in | This paper | See ‘Materials and methods,’ ‘Animals’ | |
| Cell line (H. sapiens, female) | HEK293T | Cell Bank/Stem Cell Bank, CAS | SCSP-502 | |
| Cell line (H. sapiens, female) | Nav1.2-stably expressing HEK293T | Lab of ICE Bioscience Inc | ||
| Cell line (H. sapiens, female) | Nav1.6-stably expressing HEK293T | Lab of ICE Bioscience Inc | ||
| Cell line (Cricetulus griseus, female) | Nav1.1-stably expressing CHO | Lab of ICE Bioscience Inc | ||
| Cell line (C. griseus, female) | Nav1.4-stably expressing CHO | Lab of ICE Bioscience Inc | ||
| Cell line (C. griseus, female) | Nav1.5-stably expressing CHO | Lab of ICE Bioscience Inc | ||
| Antibody | Anti-human PRRT2 (rabbit polyclonal) | Atlas Antibodies | Cat#:HPA014447; RRID:AB_1855786 | WB (1:10,000) |
| Antibody | Anti-mouse PRRT2 (rabbit polyclonal) | Wiiget Biotech | Cat#: Rp3246 | WB (1:2000) |
| Antibody | Anti-Nav1.2 (rabbit polyclonal) | Alomone Labs | Cat#:ASC-002; RRID:AB_2040005 | WB (1:2000) |
| Antibody | Anti-Nav1.1 (rabbit polyclonal) | Alomone Labs | Cat#:ASC-001; RRID:AB_2040003 | WB (1:2000) |
| Antibody | Anti-HA-Tag (rabbit monoclonal) | Cell Signaling Technology | Cat#:3724S; RRID:AB_1549585 | WB (1:1000) |
| Antibody | Anti-ATP1B2 (rabbit monoclonal) | Abcam | Cat#:ab185210 | WB (1:10,000) |
| Antibody | Anti-GAPDH-HRP (mouse monoclonal) | KangChen Bio-tech | Cat#:KC-5G5; RRID:AB_2631280 | WB (1:5000) |
| Antibody | Anti-HA nanobody magnetic beads (Alpaca nanobody) | AlfaLifeBio | Cat#:KTSM1335 | Co-IP (30 uL/test) |
| Antibody | Anti-Flag magnetic beads (mouse monoclonal) | Bimake | Cat#:B26102 | Co-IP (30 uL/test) |
| Antibody | V5-Trap magnetic beads (Alpaca nanobody) | Chromotek | Cat#:v5tma; RRID:AB_2868498 | Co-IP (30 uL/test) |
| Recombinant DNA reagent | pCAGIG (plasmid) | Addgene | Cat#:11159; RRID:Addgene_11159 | |
| Recombinant DNA reagent | pCAG-Flag-Nav1.2 | Pan et al., 2019; PMID:30765605 | Huai-Zong Shen’s Laboratory | |
| Recombinant DNA reagent | pCAG-Flag-Nav1.1 | Pan et al., 2021; PMID:33712547 | Huai-Zong Shen’s Laboratory | |
| Sequence-based reagent | sgRNA | This paper | 5’-TCTCCCACAGTGTATAAGTG-3’ | |
| Commercial assay or kit | Seamless Cloning kit | Beyotime, | Cat#:D7010 | |
| Chemical compound, drug | n-Dodecyl β-D-maltoside | Anatrace | Cat#:D310 | |
| Software, algorithm | Quantity One 1-D analysis | Bio-Rad | RRID:SCR_014280 | |
| Software, algorithm | Graphpad Prism (v8.4.3) | GraphPad Software | RRID:SCR_002798 | |
| Software, algorithm | Clampfit (v10.7.0.3) | Molecular Devices | RRID:SCR_011323 | |
| Software, algorithm | Fitmaster (v2x92) | HEKA | RRID:SCR_016233 |
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PRRT2 as an auxiliary regulator of Nav channel slow inactivation
eLife 14:RP109327.
https://doi.org/10.7554/eLife.109327.4
