An interaction between the III-IV linker and CTD in NaV1.5 confers regulation of inactivation by CaM and FHF

Figure 1.. Putative interaction site between CTD and III-IV linker. (A) Schematic of a VGSC displaying the location of the amino acids involved in the putative interaction site (numbering per NaV1.5). VSD refers to voltage sensor domain. (B) Structure of NaVPas (PDB accession no. 5X0M) as seen from the membrane. The putative interaction site is displayed in the zoomed-in images. (C) Sequence homology among different subtypes of human VGSCs and NaVPas surrounding the amino acids involved in the putative interaction, which are marked in red.

Figure S1.. Overexpression of β1 subunit does not alter the persistent currents. (A and B) Summary data for persistent currents in K1493E and E1784K in the presence or absence of β1 subunit.

Figure S2.. Conductance-voltage curves for WT and mutant VGSCs from Fig. 3, B and C. Normalized conductance (G/GMAX) values are plotted as a function of voltage. Summary of the activation properties are displayed in Table S1.

Figure 2.. FGF12B and FGF13B, but not CaM, are effective in rescuing persistent current in the Δ1885 mutant NaV1.5 channels. (A) Structure of the NaV1.5 CTD overlaid on NaVPas, with a zoomed-in view of the CTD (sky blue) along with CaM (red) and FGF13 (beige). The truncated protein in the Δ1885 mutant is shown in black. Below is the structure of the NaV1.5 CTD (blue), displaying the truncated portion in black, and the critical site for FHF binding in red (H1849), and the structure of FHF displaying the amino acid (R57) critical for the interaction with VGSC CTDs, shown in red. (B) Exemplar traces displaying the persistent currents in WT and mutant NaV1.5 channels. (C) Quantified data from WT and mutant NaV1.5 channels. *, P < 0.05. CTL refers to "control," i.e., no additional protein expressed.

Figure S3.. Effects of Δ1885 mutation on IV relation in VGSCs. Exemplar traces (A) and the I-V relationship (B) in WT and Δ1885 VGSCs from Fig. 2. pA/pF, picoamperes per picofarad.

Figure 3.. Mutations at the putative interaction site lead to an increased persistent current. (A and C) Exemplar traces displaying the persistent currents in WT and mutant VGSCs. (B and D) Quantified data of persistent currents from WT and mutant VGSCs. Gray column with black outline in D represents the WT data from B (common for both, with graphs on a different scale). *, P < 0.05.

Figure S4.. Effects of mutations at putative interaction site on IV relation in VGSCs. Exemplar traces (A) and the I-V relationships (B and C) in WT and mutant VGSCs from Fig. 3.

Figure S5.. Persistent currents in mutations outside the putative interaction site. (A and B) Summary data for the effects of K1492E mutation on persistent currents in the WT and E1784K NaV1.5 backgrounds. The WT, K1493E, and E1784K data are replicated from Fig. 3, C and D. Summary data for the effects of CaM and FGF12 on persistent currents in K1505N (C) and K1493E (D). *, P < 0.05.

Figure 4.. Mutations at the putative interaction site lead to hyperpolarized shift in steady-state inactivation. (A and C) Steady-state inactivation curves from WT and mutant channels. (B and D) Quantified data of V1/2 of inactivation from WT and mutant channels. Gray column with black outline in D represents the WT data from B (common for both). *, P < 0.05.

Figure S6.. Effects of Δ1885 and mutations at putative interaction site on Inactivation time constant. Summary data for the inactivation time constants in WT and mutant VGSCs from Fig. 2 A and Fig. 3, B and C. *, P < 0.05.

Figure 5.. CaM and FHF modulation of the persistent current is dependent on an intact putative interaction between the III-IV linker and the CTD. Black represents control, red represents addition of CaM overexpression, and blue represents addition of FGF12B overexpression in A and B). (A) Quantified data after overexpression of CaM show failure to rescue persistent currents in R1913H+K1493E. (B) CaM and FHF overexpression failed to rescue persistent currents for mutations on either side of the interaction site after overexpression of CaM and FGF12B. Gray columns with black outline in B represent the control data from WT and mutant channels from Fig. 3. *, P < 0.05.

Figure 6.. Mutations at the putative interaction site alter persistent currents in NaV1.6. (A) Sequence homology at the putative interaction sites on the III-IV linker and CTD in NaV1.5 and NaV1.6 channels. (B) Exemplar traces displaying the persistent current in WT and mutant channels of NaV1.6. (C) Steady-state inactivation curves from WT and mutant channels. (D) Quantified data of persistent current in WT and mutant NaV1.6 channels. *, P < 0.05.