Functional Consequences of the SCN5A-p.Y1977N Mutation within the PY Ubiquitylation Motif: Discrepancy between HEK293 Cells and Transgenic Mice

Figures

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  Figure 1. The SCN5A-p.Y1977N mutation abolishes the interaction between Nav1.5 and Nedd4-2 and Nedd4-2 ubiquitylation of Nav1.5. (A) Schematic representation of the four fusion proteins used for the pull-down experiments described in (B). PPSY (proline-proline-serine-tyrosine) represents the wild-type (WT) sequence of the PY-motif of Nav1.5, while PPSA and PPSN represent mutations of the tyrosine (Y) of the PY-motif in alanine (A) and asparagine (N), respectively. (B) Western blots of pull-down fractions performed on HEK293 lysates against Nedd4-2. The bottom panel shows a Ponceau staining of a representative nitrocellulose membrane, showing the presence of Glutathione S-Transferase (GST) fusion proteins for the different pull-down experiments. (C) Immunoprecipitation of Nav1.5 followed by blotting against either Nav1.5 or ubiquitin demonstrating ubiquitylation of the WT channel, but not of the YN channel, in the presence of Nedd4-2 WT. (D) Total ubiquitylated proteins from HEK293 lysates precipitated using GST-S5A fusion proteins and subsequently blotted with an antibody against Nav1.5, demonstrating a lack of WT Nav1.5 ubiquitylation in the presence of a catalytic inactive form of…

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  Figure 2. Nedd4-2-dependent regulation of sodium current is abrogated by the SCN5A-p.Y1977N mutation in HEK293 cells. Typical example of sodium current recordings obtained from HEK293 cells transiently transfected with SCN5A wild-type (WT) (A) or SCN5A-p.Y1977N (YN) (B) with or without Nedd4-2. Average sodium current-voltage relationship (C) and voltage dependence of activation and inactivation (D) for HEK293 cells transiently transfected with SCN5A WT or YN, in the presence and absence of Nedd4-2. n indicates the number of cells. Insets: voltage clamp protocols. * p < 0.05 vs. WT, # p < 0.05 vs. YN + Nedd4-2; two-way repeated measures ANOVA followed by Holm–Sidak test for post-hoc analysis.

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  Figure 3. The SCN5A-p.Y1977N mutation abolishes the Nedd4-2 mediated decrease of Nav1.5 cell surface expression. (A) HEK293 cells were transiently transfected with the indicated plasmids and whole cell lysates were blotted against Nav1.5, Nedd4-2 or actin. (B) Fluorescence intensity of Nav1.5 wild-type (WT) and Y1977N (YN) with or without Nedd4-2, was quantified with LICOR Odyssey software. Data are expressed as fold change compared to the WT or YN group. (* p < 0.05, Unpaired Student’s t-test; n corresponds to the number of experiments performed). (C) Western blot of a representative cell surface biotinylation assay in HEK293 cells transiently expressing Nav1.5 WT or YN with or without Nedd4-2. (D) Quantification of Nav1.5 protein at the cell surface in the different conditions described in panel C using LICOR Odyssey software. Data are expressed as fold change compared to the WT or YN group. (* p < 0.05, Unpaired Student’s t-test; n corresponds to the number of experiments performed).

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  Figure 4. Surface ECG measuraments and optical mapping in isolated Langendorff-perfused mice hearts. (A) Typical surface ECG recordings in wild-type (WT) and Scn5a-p.Y1981N (YN) mice. Arrows indicate how the various ECG intevals were measured. RR intervals were defined as the interval in ms between two consecutive R waves. (B) Average data for ECG parameters. No differences were observed between WT and YN mice (n indicates number of mice). (C) Typical left ventricular (LV) activation maps from an isolated WT and YN mouse heart paced at a cycle length of 120 ms.(D) Average LV longitudinal (CVlong) and LV transversal (CVtrans) conduction velocity and LV activation time in WT and YN hearts. (E) Typical examples of LV activation maps from an isolated WT and YN mouse heart during stimulation at a cycle length of 150 ms. (F) Average values for action potential duration (APD) at 50% and 80% repolarization (APD50 and APD80) in WT and YN hearts. n indicates number of mice.

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  Figure 5. Action potential properties in isolated wild-type and Scn5a-p.Y1981N mouse cardiomyocytes. (A) Typical examples of action potentials (APs) triggered at 2 Hz and first derivative (dV/dt) of the AP upstroke (inset) in wild-type (WT) and Scn5a-p.Y1981N (YN) mouse cardiomyocytes. (B) Average values for AP amplitude (APA), resting membrane potential (RMP), maximal upstroke velocity (Vmax; measured as the maximal dV/dt of the AP upstroke) and AP duration at 20%, 50%, and 90% repolarization (APD20, APD50, APD90) at the stimulation frequency of 2 Hz in WT (n = 10 cardiomyocytes from six mice) and YN cardiomyocytes (n = 14 from five mice).

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  Figure 6. Sodium current properties in wild-type and Scn5a-p.Y1981N cardiomyocytes. (A) Representative example of sodium current (INa) traces recorded in wild-type (WT) and in Scn5a-p.Y1981N (YN) cardiomyocytes during a 5-mV ascending voltage step protocol. (B) Average INa-voltage relationship (n = 8 cardiomyocytes from six WT mice and n = 7 cardiomyocytes from four YN mice), (C) voltage dependence of activation and inactivation (n = 8 cardiomyocytes from six WT mice and n = 7 from four YN mice) and (D) time course of recovery from inactivation (n = 6 cardiomyocytes from four WT mice and n = 6 cardiomyocytes from four YN mice) for WT and YN cardiomyocytes. Insets: voltage clamp protocols. (E) Typical late sodium current (INaL) traces recorded using a descending ramp protocol (upper panel) in WT and YN cardiomyocytes before (baseline) and after application of 30 µM tetrodotoxin (TTX). TTX-sensitive currents were obtained by subtraction of the current measured in the presence of TTX from the current measured earlier in the absence of TTX. (F) Average INaL-voltage relationship for WT (n = 7 cardiomyocytes from four mice) and YN cardiomyocytes (n = 6 from five mice).