Galleano I , Harms H , Choudhury K , Khoo K , Delemotte L , Pless SA .

             sodium channel activity

                long QT syndrome 3

	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 to increase contacts with DIII-S6 and DIII–S4-S5 linker. (D) Conformation of IFM particle in its binding site after 200 ns of MD simulation. Phosphorylation of Y1495 moves the loop region of the DIII-DIV linker containing the Q1483 residue outward and the IFM particle closer to the DIII-S6. IFM side chains are highlighted in orange. (E) Free energy profile of IFM unbinding, using the distance between the center of mass (COM) of N1659 in the DIV-S5 helix and F1486 in the IFM particle as a reaction coordinate. WT phY causes a decrease in the binding energy of IFM binding.
	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 further decrease of binding energy of the IFM particle compared to WT phY and an increased equilibrium distance between F1486 and its docking site due to the break in DIII-S6 induced by the Q1476R mutation.
	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.
	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).
	Fig. S1. Schematic overview of the tPTS-based approach to generate semi-synthetic Nav1.5 channels. (A) Split intein A (CfaDnaE; orange) and B (SspDnaB M86; yellow), with N- and C- terminal split intein parts shown separately. Numbers in brackets indicate amino acid numbers of split inteins. (B) Sequence of the synthetic peptide (PSYN) corresponding to amino acids 1472 to 1502 of Nav1.5. Mutated (Q1476R and ΔK1500) or modified (Y1495) sites are highlighted; the IFM motif sequence is underlined. (C) Summary of the approach, with N- and C-terminal Nav1.5 fragments (NREC and CREC) being covalently spliced on both ends of PSYN. Numbers in brackets indicate amino acid numbers in Nav1.5.
	Fig. S2. Western blots of full-length Nav1.5 protein and semi-synthetic channel constructs using antibodies directed against Nav1.5 DI-DII-linker (A) or Nav1.5 C-terminus (B). Top: The respective antibody is schematically shown on the left with its respective epitope (hNav1.5 amino acid numbering) denoted in brackets below it. The detectable constructs (fully spliced protein, splicing educts, splicing side products) are shown in basic topology and their mass is indicated in brackets. Bottom: Western blots of whole-cell lysate from Xenopus laevis oocytes expressing the respective constructs, using a different epitope. Red arrows mark the area around 230 kDa, corresponding to the mass of full length Nav1.5 protein. Lysate from uninjected oocytes and water instead of cell lysate were used as negative controls. Note that the blots also include two constructs with mutated variants of recombinant peptide X (Q1476Rrec, ΔK1500rec).
	Fig. S3. Functional characterization of conventional WT, recombinant reconstituted and semi- synthetic Nav1.5 constructs, as well as conventional single and double mutations. (A) Representative current recordings of Xenopus laevis oocytes injected with the indicated constructs. WT = full length WT Nav1.5; WTREC = NREC + CREC + PREC; WT NM = NREC + CREC + PSYN NM (i.e. containing the Y1495F and the K1479R mutations); WT phY = NREC + CREC + PSYN phY (i.e. containing a phosphonylated tyrosine in position 1495, in addition to the K1479R mutation); Y1495E, full length Nav1.5 Y1495E single mutant; Q1476R+Y1495E, full length Nav1.5 Q1476R+Y1495E double mutant. Note that uninjected oocytes, or those injected with only NREC and CREC (N + C) did not yield currents. (B) and (D) SSI (left) and activation (right) curves of indicated constructs. (C) and (E) Summary of SSI and activation (Act.) parameters of indicated constructs, see Table 1 for details. Data shown as mean ± standard deviation in (B) - (E); n = 6-16, data was compared using unpaired two-tailed student’s t-test, ns (not significant) p > 0.05, * p ≤ 0.05, *** p ≤ 0.001, **** p < 0.0001. Note that the full-length mutant channels were missing the N1472C mutation introduced in the engineered split channels, suggesting that the observed difference in SSI between Q1476R NM and Q1476R phY is likely not an artifact due to the splice-promoting mutation.
	Fig. S4. Recovery from inactivation is accelerated by phosphorylation and the Q1476R patient mutation. (A) Representative current recordings of Xenopus laevis oocytes expressing the indicated semi-synthetic Nav1.5 constructs. Oocytes were subjected to a voltage protocol consisting of two depolarizing voltage pulses to - 20 mV with a recovery interval at - 80 mV of varying length (0.5 ms to 150 ms) in between (see schematic on the right). Only a selection of traces is shown, and some traces are cropped at the beginning for clarity. (B) Recovery of channels is plotted as a single exponential function over increasing recovery intervals. Fractional recovery is calculated as the peak current elicited by the second depolarizing voltage pulse divided by the peak current elicited by the first depolarizing voltage pulse. Recovery interval is the time between the end of the first and the start of the second depolarizing voltage pulse. (C) Tau values of channel recovery, see Table 1 for details. Data shown as mean ± standard deviation; n = 5-7, data was compared using unpaired two-tailed student’s t-test, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p < 0.0001.
	Fig. S5. Phosphorylation and disease mutations can affect pharmacological sensitivity of Nav1.5 towards the anti-arrhythmic drug ranolazine. (A) Concentration response curves of WT, Q1476R and ΔK1500 constructs in response to a 20 Hz pulse train stimulation in presence of the antiarrhythmic drug ranolazine (structure shown in left panel). P50/P1 values were normalized to range from 0 to 1. (B) IC50 values obtained for ranolazine data shown in (A). The IC50 is significantly increased by phosphorylation only in WT and ΔK1500, but not in Q1476R constructs. Data shown as mean ± standard deviation; n = 6-8, data was compared using unpaired two-tailed student’s t-test, ns (not significant) p > 0.05, * p ≤ 0.05, ** p ≤ 0.01.

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