Ted similar shifts in Vrev indicating no variation in their permeability ratio (figure 5 B). Sensitivity to calcium. Reducing internal calcium concentration from 1023 to 1026 M suppressed 95.563.5 of WT TRPM4 activity (figure 5 C). A similar rapid and reversible decrease was observed for all mutants, (figure 5 D), indicating that mutants conserve the TRPM4 sensitivity to calcium. Sensitivity to voltage. The normalized open probability (NPo) for each mutant was estimated during ramp protocols, considering that the single channel conductance is linear in all cases (see figure 4). NPo was estimated in function of voltage by transforming the whole-cell current-voltage relationship (I/V) to an NPo/V curve using the relation NPo = I/gV (figure 5 E). The curve was then fitted to a Boltzman equation and voltage for half maximal activation (V1/2) was determined (figure 5 F). V1/2 wasTRPM4 Mutations in Brugada SyndromeFigure 2. Electrophoregrams of 3 missense mutations and FCCP chemical information localization of TRPM4 mutations (A ). Electrophoregrams of 3 mutations. W for A/T heterozygosity, and Y for C/T. (D): Localization of TRPM4 mutations resulting in conduction Nafarelin chemical information blocks (yellow), Brugada syndrome (blue) or both (green). doi:10.1371/journal.pone.0054131.gsignificantly increased for p.Pro779Arg compared to WT, while other mutants did not exhibited significant changes. Activation time. Activation time of the current was determined using a pulse protocol from Vm = 0 to +80 mV (figure S1 A). No significant differences were seen between mutants and WT (figure S1 B). Channel expression was evaluated in the inside-out configuration by estimating the maximal number of channels opened at Vm = +40 mV with 1023 M [Ca2+]i. As shown in figure S2, it was observed a significant decrease in the number of active channels for p.Pro779Arg compared to WT and as mentioned before, no active channels were detected for p.Lys914X. Other mutants did not shown significant variation with WT.Total and cell surface expression of TRPM4 mutant channels. To test whether the BrS mutations altered the Channel configuration. detection in the inside-outthe core glycosylated form (lower band) of total and surface expressions whereas the p.Leu1075Pro had a significant surface expression increase.DiscussionHere, we present a series of genetic variants found in a large cohort of spontaneous or drug-challenged type 1 BrS patients. Among the 14 TRPM4 variants, 3 were considered as polymorphisms. By contrast, the 11 remaining variants are presumably pathogenic mutations. Among these 11 mutations, 5 are totally absent from the very large control 
cohorts (more than 6 500 individuals), whereas 4 others have a statistically higher prevalence in the BrS cohort than in the control cohorts. As an autosomal condition with incomplete penetrance, it is not surprising that genetic variants resulting in BrS or predisposing to BrS might be found in large control cohorts. It is important to note that the size of the BrS and control cohorts allow us to use statistical tests to assess a difference of variant prevalence between both groups. The variants p.A432T and p.G844D were previously found in families with autosomal dominant cardiac conduction blocks [16]. None of the mutation carriers in the conduction block families had (even retrospectively) an ECG suggestive of a BrS [16]. Interestingly, in this series of 20 BrS cases with TRPM4 mutations or predisposing factors, 18 patients had a conduction block. The 2 patients wit.Ted similar shifts in Vrev indicating no variation in their permeability ratio (figure 5 B). Sensitivity to calcium. Reducing internal calcium concentration from 1023 to 1026 M suppressed 95.563.5 of WT TRPM4 activity (figure 5 C). A similar rapid and reversible decrease was observed for all mutants, (figure 5 D), indicating that mutants conserve the TRPM4 sensitivity to calcium. Sensitivity to voltage. The normalized open probability (NPo) for each mutant was estimated during ramp protocols, considering that the single channel conductance is linear in all cases (see figure 4). NPo was estimated in function of voltage by transforming the whole-cell current-voltage relationship (I/V) to an NPo/V curve using the relation NPo = I/gV (figure 5 E). The curve was then fitted to a Boltzman equation and voltage for half maximal activation (V1/2) was determined (figure 5 F). V1/2 wasTRPM4 Mutations in Brugada SyndromeFigure 2. Electrophoregrams of 3 missense mutations and localization of TRPM4 mutations (A ). Electrophoregrams of 3 mutations. W for A/T heterozygosity, and Y for C/T. (D): Localization of TRPM4 mutations resulting in conduction blocks (yellow), Brugada syndrome (blue) or both (green). doi:10.1371/journal.pone.0054131.gsignificantly increased for p.Pro779Arg compared to WT, while other mutants did not exhibited significant changes. Activation time. Activation time of the current was determined using a pulse protocol from Vm = 0 to +80 mV (figure S1 A). No significant differences were seen between mutants and WT (figure S1 B). Channel expression was evaluated in the inside-out configuration by estimating the maximal number of channels opened at Vm = +40 mV with 1023 M [Ca2+]i. As shown in figure S2, it was observed a significant decrease in the number of active channels for p.Pro779Arg compared to WT and as mentioned before, no active channels were detected for p.Lys914X. Other mutants did not shown significant variation with WT.Total and cell surface expression of TRPM4 mutant channels. To test whether the BrS mutations altered the Channel configuration. detection in the inside-outthe core glycosylated form (lower band) of total and surface expressions whereas the p.Leu1075Pro had a significant surface expression increase.DiscussionHere, we present a series of genetic variants found in a large cohort of spontaneous or drug-challenged type 1 BrS patients. Among the 14 TRPM4 variants, 3 were considered as polymorphisms. By contrast, the 11 remaining variants are presumably pathogenic mutations. Among these 11 mutations, 5 are totally absent from the very large control cohorts (more than 6 500 individuals), whereas 4 others have a statistically higher prevalence in the BrS cohort than in the control cohorts. As an autosomal condition with incomplete penetrance, it is not surprising that genetic variants resulting in BrS or predisposing to BrS might be found in large control cohorts. It is important to note that the size of the BrS and control cohorts allow us to use statistical tests to assess a difference of variant prevalence between both groups. The variants p.A432T and p.G844D were previously found in families with autosomal dominant cardiac conduction blocks [16]. None of the mutation carriers in the conduction block families had (even retrospectively) an ECG suggestive of a BrS [16]. Interestingly, in this series of 20 BrS cases with TRPM4 mutations or predisposing factors, 18 patients had a conduction block. The 2 patients wit.