Bands obtained after cell surface protein biotinylation from 6 independent experiments. Densitometry analysis of the bands (Fig. 5B and C) did not reveal statistically significant differences between WT and I890T cells (Nav1.5 I890T/WT was 1.1160.14, n = 6). Taken collectively, our data indicate that the I890T mutation causes a loss-of-function of Nav1.5 current by the modification of the biophysical properties intrinsic to channel activity, rather than by impaired expression at the plasma membrane.In silico Models of the Pore Region of WT and I890T DII of Nav1.We performed a sequence alignment between DII of Nav1.5 channel and the corresponding sequence of NavAb channel (Fig. 6A, upper panel) and built models for the S5 6 region of the human channel using protein structure prediction models. Figure 6B shows the prediction obtained using the CPHmodels tool [20]. The loop S5 6 in our model had a longer, flexible turret loop before the P1-helix. I890 resided within the second turn of the P1-helix, buried among the turret loop, the selectivity filter and the P2-helix.The structure of the S5 6 loop was not apparently affected by the introduction of the I890T mutation (not shown).DiscussionFigure 4. I890T does not affect the time course of inactivation, slow inactivation, or recovery from inactivation. (A) Experimental data obtained for the current-voltage relationship (Fig. 2) was used to determine inactivation time constants in the voltage range between 240 and 20 mV. Current decay after the peak INa was Methionine enkephalin price fitted to a monoexponential function (from 240 to 225 mV) and a bi-exponentialIn the present work we identified a novel SCN5A mutation in a patient diagnosed with BrS. The same mutation was found in three other family members, two of whom presented signs of potential arrhythmogenicity. Complete genetic 23977191 analysis of the SCN5A gene revealed that the younger sister, who did not present any cardiac abnormalities, carried a common polymorphism (p.H558R), inherited from the father. This variation was not foundNovel Nav1.5 Pore Mutation I890T Causes BrSFigure 5. Membrane expression of Nav1.5 channel is not affected by I890T. Western blot detection of Nav1.5 and Na+/K+ 
ATPase proteins performed after cell surface biotinylation from WT and I890T cells. (A) Image shows the bands obtained for Nav1.5 and Na+/K+ ATPase, in 6 independent experiments from cells expressing either WT or I890T. Position of the markers is shown on the left side. Numbers correspond to each experiment. (B) Bar graph depicts the Nav1.5 intensity values normalized by the Nav1.5 WT intensity values (n = 6). Nav1.5 I890T intensity values were previously corrected by multiplying the raw Tetracosactide chemical information integrated density values by the ratio between the WT and I890T Na+/K+ ATPase integrated density values. Note that for experiment number 5, the ratio between I890T and WT was calculated from the average of the intensity values obtained for the two samples of each condition. (C) Bar graph shows the average intensity values (expressed as mean 6 SE) obtained in (B). doi:10.1371/journal.pone.0053220.gin any of the other mutation carriers. One of the most intriguing features of BrS is the marked phenotypic variability. Clinical phenotype of individuals that carry SCN5A mutations may range from asymptomatic to SCD [26]. It has been postulated that modifying factors, such as genetic background and environment, influence the clinical phenotype of BrS patients [27,28]. Several factors may explain the absence.Bands obtained after cell surface protein biotinylation from 6 independent experiments. Densitometry analysis of the bands (Fig. 5B and C) did not reveal statistically significant differences between WT and I890T cells (Nav1.5 I890T/WT was 1.1160.14, n = 6). Taken collectively, our data indicate that the I890T mutation causes a loss-of-function of Nav1.5 current by the modification of the biophysical properties intrinsic to channel activity, rather than by impaired expression at the plasma membrane.In silico Models of the Pore Region of WT and I890T DII of Nav1.We performed a sequence alignment between DII of Nav1.5 channel and the corresponding sequence of NavAb channel (Fig. 6A, upper panel) and built models for the S5 6 region of the human channel using protein structure prediction models. Figure 6B shows the prediction obtained using the CPHmodels tool [20]. The loop S5 6 in our model had a longer, flexible turret loop before the P1-helix. I890 resided within the second turn of the P1-helix, buried among the turret loop, the selectivity filter and the P2-helix.The structure of the S5 6 loop was not apparently affected by the introduction of the I890T mutation (not shown).DiscussionFigure 4. I890T does not affect the time course of inactivation, slow inactivation, or recovery from inactivation. (A) Experimental data obtained for the current-voltage relationship (Fig. 2) was used to determine inactivation time constants in the voltage range between 240 and 20 mV. Current decay after the peak INa was fitted to a monoexponential function (from 240 to 225 mV) and a bi-exponentialIn the present work we identified a novel SCN5A mutation in a patient diagnosed 
with BrS. The same mutation was found in three other family members, two of whom presented signs of potential arrhythmogenicity. Complete genetic 23977191 analysis of the SCN5A gene revealed that the younger sister, who did not present any cardiac abnormalities, carried a common polymorphism (p.H558R), inherited from the father. This variation was not foundNovel Nav1.5 Pore Mutation I890T Causes BrSFigure 5. Membrane expression of Nav1.5 channel is not affected by I890T. Western blot detection of Nav1.5 and Na+/K+ ATPase proteins performed after cell surface biotinylation from WT and I890T cells. (A) Image shows the bands obtained for Nav1.5 and Na+/K+ ATPase, in 6 independent experiments from cells expressing either WT or I890T. Position of the markers is shown on the left side. Numbers correspond to each experiment. (B) Bar graph depicts the Nav1.5 intensity values normalized by the Nav1.5 WT intensity values (n = 6). Nav1.5 I890T intensity values were previously corrected by multiplying the raw integrated density values by the ratio between the WT and I890T Na+/K+ ATPase integrated density values. Note that for experiment number 5, the ratio between I890T and WT was calculated from the average of the intensity values obtained for the two samples of each condition. (C) Bar graph shows the average intensity values (expressed as mean 6 SE) obtained in (B). doi:10.1371/journal.pone.0053220.gin any of the other mutation carriers. One of the most intriguing features of BrS is the marked phenotypic variability. Clinical phenotype of individuals that carry SCN5A mutations may range from asymptomatic to SCD [26]. It has been postulated that modifying factors, such as genetic background and environment, influence the clinical phenotype of BrS patients [27,28]. Several factors may explain the absence.
