Of symptoms in the younger sister. Young age and being a female are factors that diminish risk of arrhythmogenesis in BrS patients. In addition, the younger sister carries the polymorphism p.H558R which has been identified as a palliative factor in the pathological effects of BrS associated mutations [29,30]. Still, this polymorphism was not found in themother who, despite having suffered syncope, was not diagnosed with BrS. It is evident from these data that a combination of modifying factors is determinant of the clinical phenotype, especially when the functional effect of the pathogenic mutation is 15900046 mild. The c.2669 T.C nucleotidic change produces an amino acidic variation of an isoleucine-to-threonine in position 890 (p.Iso890Thr, p.I890T), localized in the P-loop of the domain II of the Nav1.5 channel (Fig. 6C). Sequence alignment demonstrated that I890 is conserved within the members of the voltage-gated sodium channel a-subunit family as well as in Nav1.5 channels ofNovel Nav1.5 Pore Mutation I890T Causes BrSFigure 6. I890 is a conserved aminoacid, located in the intramembrane pore region of Nav1.5 DII. (A) Sequence alignment of the pore modules of human Nav1.5 channel (DII) and NavAb. Identical aminoacids are Tubastatin-A highlighted in grey. Isoleucine-890 is marked with a dark box. Similar aminoacids are included inside light 
boxes and dots identify insertions (lower panel). Sequence alignment of human voltage-gated sodium channel a-subunit family members and of Nav1.5 channels of different species, upper and middle 58-49-1 panels, respectively. 
The position of the first amino acid of each sequence is indicated on the left side, and the reference for each protein according to Uniprot is shown at the right side. (B) Partial view of the CPHmodel showing the pore module of DII of Nav1.5 channel (in green), based on the coordinates of NavAb channel (in red). I890Nav1.5 and T169NavAb are located in the middle of P1-helix and highlighted in blue and magenta, respectively. View from the interior side of the pore. (C) Nav1.5 channel scheme. The relative position of the I890T mutation in the S5 6 loop of domain II (DII) is indicated with an arrow. doi:10.1371/journal.pone.0053220.gother species (Fig. 6A, middle and lower panels), indicating that a hydrophobic residue is essential at this position. A correlation between the region of the Nav1.5 channel where mutations are located and the severity of the clinical phenotype has not been well established. Meregalli et al. [31] published a systematic study showing a correlation between the type of mutation and the changes produced in Nav1.5 currents. A broader study would most likely give light to this intriguing subject. Unfortunately, this type of study is difficult to achieve mainly due to the fact that only a small percentage of BrS related mutationsare studied functionally. We have performed an exhaustive compilation of reported mutations related to BrS located in the pore regions of Nav1.5 channel (Table S1). Out of the 86 mutations identified in these regions, functional studies are documented only for 18 of them. The present work is the first reported functional study of a pore mutation in DII of Nav1.5 associated with BrS. Our electrophysiological studies in transfected HEK cells showed that the mutation caused a significant decrease in current density compared to WT. In addition, the activation curve of theNovel Nav1.5 Pore Mutation I890T Causes BrSI890T currents was shifted to more positive potential.Of symptoms in the younger sister. Young age and being a female are factors that diminish risk of arrhythmogenesis in BrS patients. In addition, the younger sister carries the polymorphism p.H558R which has been identified as a palliative factor in the pathological effects of BrS associated mutations [29,30]. Still, this polymorphism was not found in themother who, despite having suffered syncope, was not diagnosed with BrS. It is evident from these data that a combination of modifying factors is determinant of the clinical phenotype, especially when the functional effect of the pathogenic mutation is 15900046 mild. The c.2669 T.C nucleotidic change produces an amino acidic variation of an isoleucine-to-threonine in position 890 (p.Iso890Thr, p.I890T), localized in the P-loop of the domain II of the Nav1.5 channel (Fig. 6C). Sequence alignment demonstrated that I890 is conserved within the members of the voltage-gated sodium channel a-subunit family as well as in Nav1.5 channels ofNovel Nav1.5 Pore Mutation I890T Causes BrSFigure 6. I890 is a conserved aminoacid, located in the intramembrane pore region of Nav1.5 DII. (A) Sequence alignment of the pore modules of human Nav1.5 channel (DII) and NavAb. Identical aminoacids are highlighted in grey. Isoleucine-890 is marked with a dark box. Similar aminoacids are included inside light boxes and dots identify insertions (lower panel). Sequence alignment of human voltage-gated sodium channel a-subunit family members and of Nav1.5 channels of different species, upper and middle panels, respectively. The position of the first amino acid of each sequence is indicated on the left side, and the reference for each protein according to Uniprot is shown at the right side. (B) Partial view of the CPHmodel showing the pore module of DII of Nav1.5 channel (in green), based on the coordinates of NavAb channel (in red). I890Nav1.5 and T169NavAb are located in the middle of P1-helix and highlighted in blue and magenta, respectively. View from the interior side of the pore. (C) Nav1.5 channel scheme. The relative position of the I890T mutation in the S5 6 loop of domain II (DII) is indicated with an arrow. doi:10.1371/journal.pone.0053220.gother species (Fig. 6A, middle and lower panels), indicating that a hydrophobic residue is essential at this position. A correlation between the region of the Nav1.5 channel where mutations are located and the severity of the clinical phenotype has not been well established. Meregalli et al. [31] published a systematic study showing a correlation between the type of mutation and the changes produced in Nav1.5 currents. A broader study would most likely give light to this intriguing subject. Unfortunately, this type of study is difficult to achieve mainly due to the fact that only a small percentage of BrS related mutationsare studied functionally. We have performed an exhaustive compilation of reported mutations related to BrS located in the pore regions of Nav1.5 channel (Table S1). Out of the 86 mutations identified in these regions, functional studies are documented only for 18 of them. The present work is the first reported functional study of a pore mutation in DII of Nav1.5 associated with BrS. Our electrophysiological studies in transfected HEK cells showed that the mutation caused a significant decrease in current density compared to WT. In addition, the activation curve of theNovel Nav1.5 Pore Mutation I890T Causes BrSI890T currents was shifted to more positive potential.