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

Fig. 2. Insertion of recombinantly expressed peptides into the DIII–IV linker of NaV1.5.

Fig. 2

a Schematic presentation of the strategy to reconstruct full-length NaV1.5 from recombinantly expressed N-/C-terminal fragments (N and C) and a recombinantly expressed peptide corresponding to amino acids 1472 to 1502 of the NaV1.5 DIII–DIV linker (X) in Xenopus laevis oocytes. Inteins A (CfaDnaE) and B (SspDnaBM86) are indicated by square and round symbols, respectively. Note that we cannot exclude the possibility that splicing takes place at a different subcellular location than depicted here. b Representative sodium currents in response to sodium channel activation protocol (see methods; only voltage steps from −50 to +10 mV in 10 mV steps are displayed), demonstrating expression of functional NaV1.5 only in the presence of all three components (N + C + X), along with WT and N1472C channels (the latter mutation was introduced to create an optimized splice site for intein A. c Immunoblots verifying the presence of fully spliced NaV1.5 only when all three components (N + C + X) were co-expressed (using antibody against NaV1.5 C-terminus). NaV1.5 band was not detected when one component was missing (left blot) or when non-splicing mutation (N + C + X mut.) was introduced to prevent splicing (right blot; see Supplementary Fig. 2). Black arrows indicate band positions of the respective constructs (Actual MW of constructs: WT, 227 kDa; C-construct, 79 kDa; C + X, 65 kDa; C-terminal cleavage product, C*, 58 kDa). Note that X and XREC refer to XNav1.5REC in this panel. d Steady-state inactivation and conductance–voltage (G–V) relationships for functional constructs tested. e Comparison of values for half-maximal (in)activation (V50) (values are displayed as mean + /– SD; WT, n = 12; N1472C, n = 14; N + C + XNav1.5REC, n = 16). Significant differences were determined by one-way ANOVA with a Tukey post-hoc test. *p < 0.03 (WT vs. N1472C, p = 0.014; WT vs. N + C + XNav1.5REC, p = 0.012). Source data are provided as a Source data file.