PRRT2 regulates slow inactivation of Nav1.2 channels.

(A) Representative immunoblots showing PRRT2 protein expression in Nav1.2-stably expressing HEK293T cells transfected with constructs encoding either EGFP or mouse PRRT2 (mPRRT2). MW, molecular weight. (B) Schematic of whole-cell recording (left) and representative traces of sodium currents evoked by a brief depolarization (right). The arrow indicate sodium current decay due to fast inactivation. Scale bar, 1 nA by 2 ms. (C) Decay time constants of sodium currents in response to 20-ms depolarization (EGFP: n = 13 cells, mPRRT2: n = 13 cells). (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 recover 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 progressive longer depolarization (EGFP: n = 6 cells, mPRRT2: n = 6 cells). (G) The effects of PRRT2 on the recovery of Nav1.2 channels from slow-inactivated state induced by 5-s depolarization (EGFP: n = 6 cells, PRRT2: n = 6 cells). Data were 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.

PRRT2 promotes steady-state slow 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 by 2 ms. (B and C) The effects of PRRT2 on voltage-dependent steady-state slow inactivation of Nav1.2 channels (B) (EGFP: n = 11 cells, mPRRT2: n = 13 cells). mPRRT2, mouse PRRT2. Data were presented as mean ± s.e.m. Two-way ANOVAs were used in (B) to determine the statistical significance of main effects of group. ****P < 0.0001.

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 truncates. 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) The effects of PRRT2 truncates 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) The effects of PRRT2 truncates 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 were presented as mean ± s.e.m. The main effect of group was assessed using two-way ANOVAs (B, C, E and F). **P < 0.01, ****P < 0.0001, n.s., not significant.

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 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). Identity is calculated by using 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 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 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 were presented as mean ± s.e.m. The main effect of group was assessed using two-way ANOVAs (C, D, F and G). **P < 0.01, ****P < 0.0001, n.s., not significant.

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 carboxyl terminus of these proteins are underlined. REL, re-entrant loop. TM, transmembrane domain. (B) Schematic showing the phylogenetic relationship between members of 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 slowinactivated states induced by 5-s depolarization (EGFP: n = 10 cells, TRARG1: n = 14 cells, TMEM233: n = 12 cells, PRRT2: n = 12 cells). Data were presented as mean ± s.e.m. The main effect of group was assessed using two-way ANOVAs (C and D). **P < 0.01, ****P < 0.0001, n.s., not significant.

Uniform influence of PRRT2 on slow inactivation across human Nav isoforms.

(A) Protocol for examining the entry of Nav channels into slow-inactivated state. (B-D) The effects of human PRRT2 (hPRRT2) on the entry of Nav1.1, Nav1.4 and Nav1.5 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). (E) Protocol for examining the recovery of Nav channels from slow-inactivated state. (F-H) The effects of human PRRT2 on the recovery of Nav1.1, Nav1.4 and Nav1.5 channels from slow-inactivated state induced by 5-s depolarization. In (F), analyses for Nav1.1 (EGFP: n = 10 cells, hPRRT2: n = 12 cells); In (G), analyses for Nav1.4 (EGFP: n = 11 cells, hPRRT2: n = 11 cells); In (H), analyses for Nav1.5 (EGFP: n = 16 cells, hPRRT2: n = 15 cells). Data were presented as mean ± s.e.m. The main effect of group was assessed using two-way ANOVAs (B-D and F-H). ****P < 0.0001.

Absence of effect of PRRT2 on Kv1.4 channel inactivation.

(A) Illustration showing the whole-cell voltage-clamp recording in Kv1.4 stably expressing HEK293T cells (upper) and representative traces of potassium currents evoked by a depolarization at 40 mV (bottom). Scale bar, 1 nA by 50 ms. (B) The effects of human PRRT2 on the entry of Kv1.4 channels into inactivated state (EGFP: n = 9 cells, hPRRT2: n = 8 cells). (C) The effects of human PRRT2 on the recovery of Kv1.4 channels from inactivated states induced by 5-s depolarization (EGFP: n = 8 cells; hPRRT2: n = 7 cells). Data were presented as mean ± s.e.m. The main effect of group was assessed using two-way ANOVAs (B and C). n.s., not significant.

Intermolecular interaction between PRRT2 and Nav channels in vitro.

(A and B) Schematic diagram showing the co-immunoprecipitation for detecting human PRRT2-Nav1.2 interaction. 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 and E) Co-immunoprecipitation assay for potential interaction 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 and E). (F) Diagram for HA-tagged truncation of human PRRT2 (PRRT2(250–340)), in which the amino terminus of PRRT2 was deleted. (G and H) Co-immunoprecipitation assay for potent interaction 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 and H). (I and J) Co-immunoprecipitation assay for potential interaction 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 and 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.

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, protospaceradjacent motif (NGG). ssDNA, single-strand 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 were 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.

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 (right). The arrow indicates an axonal bled. Scale bar, 20 μm. (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). (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 by 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 were 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 were presented as mean ± s.e.m. Two-tailed, unpaired Student’s t-test was used in (B and 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.

Protein levels of Nav1.2 are unchanged in brain tissue from Prrt2-mutant mice.

(A and B) Immunoblots (A) and quantifications (B) of Nav1.2 proteins in brain tissue of wildtype (WT) and Prrt2-mutant mice (n = 3 mice). Data were 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.