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. 2020 May 8;11:2284. doi: 10.1038/s41467-020-16208-6

Fig. 3. Insertion of synthetic peptides into NaV1.5.

Fig. 3

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.