Fig 2: Insertion of synthetic peptides into NaV1.5.a Schematic of strategy to reconstruct full-length NaV1.5 from recombinantly expressed N-/C-terminal fragments (N and C) and a synthetic peptide (XNav1.5SYN) in Xenopus laevis oocytes. Inteins A (CfaDnaE) and B (SspDnaBM86) indicated by square and round symbols, respectively. Note: we cannot exclude that splicing takes place at a different subcellular location than depicted. b Peptide XNav1.5SYN sequence corresponding to amino acids replaced in the NaV1.5 DIII–DIV linker and chemical structures of native amino acids and PTM mimics (tAcK/phY) incorporated via chemical synthesis of peptide XNav1.5SYN. The N1472C (underlined) mutation was introduced to optimize splicing (Supplementary Fig. 1). c Immunoblot verifying the presence of fully spliced NaV1.5 only when peptide XNav1.5SYN was co-injected with N and C. Black arrows indicate band positions of the respective constructs (Actual MW of constructs: WT, 227 kDa; C, 79 kDa; C + X, 65 kDa; C-terminal cleavage product, C*, 58 kDa). Band at ~150 kDa is possibly a dimer or aggregate of C. d Representative sodium currents (see Methods; only voltage steps from −50 to +10 mV in 10 mV steps are displayed), demonstrating expression of functional NaV1.5 when Xenopus laevis oocytes expressing N and C constructs were injected with synthetic peptides containing non-modifiable side chains in positions 1479 and 1495 (K1479R and Y1495F, NM), tAcK1479 or phY1495 or both PTM mimics together. e Average current amplitudes recorded at −35 mV from oocytes expressing N and C constructs and injected with synthetic peptide variant NM or phY1495 depicted as a bar plot (mean + /– SD; WT, n = 8; NM, n = 9; phY, n = 8; N + C, n = 6). Currents normalized to mean currents measured from oocytes expressing the full-length WT construct. To ensure adequate control of voltage clamp, [Na+] in the extracellular recording solution was reduced (see Supplementary Fig. 3 for details). f Steady-state inactivation and conductance–voltage (G–V) relationships for PTM-modified/non-modified constructs (values displayed as mean + /– SD; N1472C, n = 15; NM, n = 10; tAcK1479, n = 21; phY1495, n = 19; tAcK1479 + phY1495, n = 14). Source data are provided as a Source data file.

Fig 3: Expression of TTXr Sodium Channels in Developing rat DRGs(A) mRNA expression levels of Scn5a, Scn9a, Scn10a, and Scn11a in DRGs of embryonic (E14–E16) and neonatal (P6) rats. Scn5a is downregulated and Scn11a upregulated (in neonatal DRGs compared with E14–E16 (n = 4, two independent experiments, shown as technical duplicates).(B) Bottom: representative traces of voltage clamp recordings of E14–E16 and P6 DRGs in the presence of 500 nM TTX. Top: voltage protocol.(C) Voltage dependence of activation of HUES6-derived (triangles, red fits, n = 15) and hiPSC-derived nociceptors (diamonds, blue fits, n = 26) is shifted toward more hyperpolarized potentials compared with rat E14–E16 DRGs (circles, black fits, n = 13 cells) and P6 DRGs (squares, black fits, n = 21 cells).(D) Voltage dependence of steady-state fast inactivation of HUES6-derived (n = 15) and hiPSC-derived nociceptors (n = 23) is shifted toward more hyperpolarized potentials compared with embryonic and neonatal rat DRGs (n = 4 of 19 cells). Data of HUES6- and hiPSC-derived nociceptors are the same as shown in Figure 2.All error bars indicate the SEM.

Fig 4: Alternative splicing of exon 6 and 6A in the SCN5A gene. (A) Agarose gel depicting the electrophoresis results following polymerase chain reaction with primer P2. A single, clear band with the expected size of 354 bp was observed. Lanes 1–3, samples of human brain cortex from the frontal lobe. (B) Electrophoresis results following digestion by restriction enzyme SacI. Products with restriction enzyme SacI generated three bands on the agarose gel, with the expected sizes of 354, 248 and 106 bp, respectively, indicating that fragments including exon 6A were digested into another two fragments, while those containing exon 6 were preserved. Lanes 1E-3E, PCR products with restriction enzyme SacI; lanes 1–3, PCR products without restriction enzyme SacI. (C) Schematic for the alternative splicing of exons 6 and 6A of the SCN5A gene. Constitutive exons are presented in gray boxes, alternatively spliced sequences are presented in solid boxes and introns are presented as solid lines. The two different pathways are presented as poly lines. M, marker.

Fig 5: a Differentiated hiPSC-derived pacemaker cells express the SAN channel HCN4 and the cardiac channels Nav1.5 and Cav1.2. b Immunofluorescence staining of hiPSC-derived ventricular-like cells and hiPSC-derived pacemaker-like cells against connexin 43 (magenta), connexin 40 (red) and connexin 45 (green). Single channel pictures. c Schematic interaction between CVB3 and hiPSC-derived pacemaker cells concerning HCN4 transport and function as it was observed in this study. Not only is CVB3 a player in general HCN4 expression but mainly disturbs HCN4 recycling via Rab7-dependent autophagy combined with elevated autophagosome formation, leading to the loss of HCN4 function