Functional cross-talk between phosphorylation and disease-causing mutations in the cardiac sodium channel Na v 1.5

Figures

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  Fig. 1.. Phosphorylation of Y1495 destabilizes docking of IFM motif into its receptor site. (A) Schematic of tPTS used to generate Nav1.5 channels that are NM (WT NM) or phosphorylated (WT phY) at the Y1495 position. Amino acids (aa) 1 to 101 of CfaDnaE (orange) are merged to the C terminus of channel fragment corresponding to Nav1.5 aa 1 to 1471 for heterologous expression as the NREC construct. The PSYN sequence corresponds to Nav1.5 aa 1472 to 1502 and is linked to the C-terminal part of CfaDnaE (aa 102 to 137, orange) at its N terminus and the N-terminal part of SspDnaBM86 (aa 1 to 11, yellow) at its C terminus. The corresponding C-terminal part of SspDnaBM86 (aa 12 to 154, yellow) is expressed as a fusion construct at the N terminus of protein fragment C (Nav1.5 aa 1503 to 2016) to form the CREC construct. (B) SSI (Left) and activation (Right) curves of WT NM and WT phY constructs, including example traces and chemical structure of the aa present in position 1495. Data shown as mean ± SD; n = 6 to 9. (C) Contact frequency of Y1495 and IFM particle residues with neighboring residues. Phosphorylation reduces the contact between Y1495 and M1487 of IFM, while it causes the IFM particle…

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  Fig. 2.. The Q1476R mutation results in a dramatically shifted SSI. (A) Representative current traces for Q1476R NM (purple) and phY (blue) constructs. (B) SSI (Left) and activation (Right) curves of indicated constructs. (C) Late currents (Upper) and inactivation rates (Lower) for indicated constructs. Data are shown as mean ± SD in B and C; n = 5 to 10; data were compared using unpaired two-tailed Student’s t test; ns (not significant) P > 0.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P < 0.0001. Note that the precise measurements of late currents in X. laevis oocytes are hampered by slow-onset, voltage-dependent endogenous currents. Therefore, our late current measurements can only serve as an estimate. (D) Contact frequency of Y1495 and IFM particle with neighboring residues. (E) Conformation of IFM particle in its binding site after 200 ns of MD simulation. Q1476R phY leads to a break in the DIII-S6 helix, leading to the destabilization of IFM binding. IFM side chains are highlighted in orange. (F) Free energy profile of IFM unbinding, using the distance between the COM of N1659 in DIV-S5 helix and F1486 in the IFM particle as a reaction coordinate. Q1476R phY results in a…

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  Fig. 3.. Phosphorylation-induced SSI shift is similar in ΔK1500 and WT. (A) Representative current traces for ΔK1500 NM (light green) and ΔK1500 phY (dark green) constructs. (B) SSI (Left) and activation (Right) curves of indicated constructs. (C) Late currents (Upper) and inactivation rates (Lower) for indicated constructs. Data are shown as mean ± SD in B and C; n = 6 to 10; data were compared using unpaired two-tailed Student’s t test; ns (not significant) P > 0.05, *P ≤ 0.05, ***P ≤ 0.001, and ****P < 0.0001. (D) Contact frequency of Y1495 and IFM particle with neighboring residues. (E) Conformation of the IFM particle in its binding site after 200 ns of MD simulation. I1485 increases its contacts with DIII-S6 residues. IFM side chains highlighted in orange. (F) Free energy profile of IFM unbinding using the distance between the COM of N1659 in the DIV-S5 helix and F1486 in the IFM particle as a reaction coordinate. The binding energy of the IFM particle for the ΔK1500 phY system is similar to that of WT phY.

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  Fig. 4.. Phosphorylation and disease mutations can affect pharmacological sensitivity of Nav1.5. (A and B) Concentration response curves of WT, Q1476R, and ΔK1500 constructs in response to a 20-Hz pulse train stimulation in presence of AADs quinidine (A) or flecainide (B), respectively (see structures in left panels). P50/P1 values were normalized to range from 0 to 1. (C) IC50 values obtained for quinidine data shown in A. The IC50 is significantly increased by phosphorylation only in WT but not in Q1476R or ΔK1500 constructs. (D) IC50 values obtained for flecainide data shown in B. IC50 values are not significantly altered by phosphorylation in any of the constructs. ns (not significant) P ≥ 0.05 and *P < 0.05. Data shown as mean ± SD; n = 5 to 9. (E) Overlay of flecainide (F) bound to rat Nav1.5 (yellow; PDB code: 6UZ0) and quinidine (Q) bound to human Nav1.5 (orange; PDB code: 6LQA).