Gosselin-Badaroudine P , Keller DI , Huang H , Pouliot V , Chatelier A , Osswald S , Brink M , Chahine M .

MT-13181 Canadian Institutes of Health Research

	Figure 1. Family pedigree, clinical evaluation, and molecular genetics.(A) The index patient (III-1) is indicated by an arrow. Individuals indicated with black squares/circles carry the mutation and a clinical phenotype (III-1, III-2, II-2). Individuals indicated with grey circles (II-3 to II-5) were clinically diagnosed with DCM, but not genotyped. Abbreviation: DCM (dilated cardiomyopathy). (B) 12-lead ECG of the index patient showing third degree AV-block with a ventricular escape rhythm and a small QRS-complex with a heart rate of 43 bpm (artefact in lead V1). (C) Non-sustained ventricular tachycardia (220 bpm) occurred at a heart rate of 130 bpm and a work load of 192 W during an exercise stress test. (D) Different DHPLC eluting profiles at 59.8°C of the PCR products of exon 6 in the index patient compared to the control. Abbreviation: DHPLC (denaturing high performance liquid chromatography). (E) A heterozygous change of arginine CGC (R) to histidine CAC (H) resulted in the missense mutation R219H. (F) Sequence alignments of the S4 of domain 1 from Na+ and K+ (Shaker B) channels in different species.
	Figure 2. Biophysical characterization of the Nav1.5/R219H DCM mutation proton current recordings.Representative current traces recorded using the cut-open oocyte technique from Nav1.5/WT (A) and Nav1.5/R219H (B) channels. Currents were elicited by depolarizing pulses from −100 mV to +60 mV, with 10 mV increments for each step. (C) The voltage dependence of steady-state activation and inactivation of WT (activation, n = 7; inactivation, n = 8) and R219H (activation, n = 8; inactivation, n = 8). Activation curves were derived from I–V curves and fitted to a standard Boltzmann equation: G (V)/G max = 1/(1+exp ((V−V1/2)/kv)), with midpoints (V1/2) is slow factors (kv) listed in Table S1. The voltage-dependence of inactivation was induced by applying conditioning pre-pulses to membrane potentials ranging from a holding potential of −150 to −20 mV for 500 ms with 10 mV increments and was then measured using a 20-ms test pulse to −30 mV for each step (see protocol in inset). The recorded inactivation data were fitted to a standard Boltzmann equation: I (V)/Imax = 1/(1+exp ((V−V1/2)/kv)), with midpoints (V1/2) is slow factors (kv) listed in Table S1. (D) Time courses of recovery from inactivation of Nav1.5/WT and Nav1.5/R219H channels. A 40 ms conditioning pre-pulse was used to monitor recovery using a 20-ms test pulse after a variable recovery interval ranging from 5 to 500 ms (see protocol in inset). A single-exponential function was used to determine the time constants of recovery.
	Figure 3. Nav1.5/R219H exhibits a pH dependent current.Panel (A) shows that varying the Ringer's extracellular pH (pHo) induced an inward current in Nav1.5/R219H–injected oocytes. An acidic Ringer's solution induced inward currents in an oocyte expressing the Nav1.5/R219H channel. The oocyte was held at −80 mV and a −140 mV test pulse was repeated every 2 s (only current responses at −140 mV are shown). The bottom panels show current traces of the experiment in (A). The inset shows the protocol, and the grey zone indicates where currents were measured as a mean of the current amplitude between 150 and 200 ms.(B) Effect of an acidic Ringer's solution in the presence of TTX in an oocyte expressing the Nav1.5/R219H mutant channel in a background in which the native cysteine in D1 had been replaced with a tyrosine (C373F) and in the presence of 1 µM TTX. This mutation in the pore region increases the TTX sensitivity of cardiac channels 60- to100-fold, as described previously [46]. (C) Currents recorded from a water-injected and oocyte expressing the Nav1.5/WT or Nav1.5/R219H channel. The oocytes were held at −80 mV and were pulsed to −140 mV. This protocol was repeated every 2 s as indicated in the inset (panel A). Acidic NMDG solutions induced a pH-dependent current in an oocyte expressing the Nav1.5/R219H channel in a C373F background in the presence of 1 µM TTX. This experiment was carried out in a Nav1.5 background in which the native cysteine in D1 was replaced with a tyrosine (C373F). (D) Acidic NMDG solutions induced a reversible current in an oocyte expressing the Nav1.5/R219H channel.
	Figure 4. Nav1.5/R219H induces an inward proton current and intracellular acidification.Xenopus oocytes expressing Nav1.5/WT or Nav1.5/R219H channel were impaled with three electrodes, one filled with an H+ resin to measure pHi, and two to clamp the oocyte at −80 mV in a Na+-free NMDG solution containing 1 µM TTX, as indicated. Typical proton current recordings (red traces) in response to different pHo value and the pHi measurement rate (bleu traces) from an oocyte expressing the Nav1.5/R219H (A) or Nav1.5/WT channel (B). Intracellular pHi values before changing solutions in experiments similar to (A) and (B) were plotted against pHo (***, p<0.001 compared to WT, n = 10–19)(C). Similar recordings were obtained with four batches of oocytes. (D) Changes in pHi after incubating oocytes expressing the Nav1.5/WT (triangles) or Nav1.5/R219H (squares) channel, or water-injected oocytes (circles) in OR3 medium at different pHo values (***, p<0.001, **; p<0.01; *, p<0.05; compared to WT, n = 7–13). pHi measurements were carried out in Ringer's solution at pHo of 7.40.
	Figure 5. Proton current-voltage relationship of the Nav1.5/R219H channel recorded in an NMDG Na+-free solution.(A) Representative proton current traces from oocytes expressing the Nav1.5/R219H channel recorded at pHo 8.40, 7.40, 6.80, and 6.00, as indicated, in response to 200 ms voltage steps ranging from −140 mV to +40 mV in 5-mV increments from a holding potential of −80 mV (the protocol is given in the centre inset), without on-line leak subtraction. The dashed line represents the zero current. For clarity, only current every 10 mV are shown. (B) Current-voltage relationship where the currents in (A) were plotted as a function of the test potential (5 mV increments), after offline linear leak subtraction. Reversal potential determined in a Na+-free NMDG solution at pHo 8.40 using voltage steps as described in (A). The pHi was measured using a pH-sensitive electrode. Similar results were obtained with four separate batches of oocytes. The inset shows the pHoand pHi values and between parentheses is the predicted values calculated using the Nernst equation. The bleu trace shows the voltage-dependent of activation (Q–V), the grey zone illustrates the transitional zone corresponding to the probability of the voltage sensor being stabilized in the outward position. (C) Correlation between the peak Na+ current measured in Ringer's solution and the proton current measured at −140 mV and pHo 4.00 (n = 31) on the same oocytes. The data were obtained from one batch of oocytes over three days. The straight line represents the linear regression of the data set and R2 is the correlation coefficient and shows the goodness of fit. Similar results were obtained with three separate batches of oocytes. (D) Proton currents measured in response to a change in pHo at −140 mV in an NMDG Na+-free solution. The currents were normalized to the currents obtained at pHo = 4.00 for each cell. The mean data (n = 5) was fitted to the Henderson-Hasselbach equation, 1/[1+exp(2.3(pHo−pKa))]. Error bars are smaller than the symbols.

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