E 3 missense mutations gave a Grantham score [19] of 89 or more. Grantham

E 3 missense mutations gave a Grantham score [19] of 89 or more. Grantham is a formula estimating difference between amino acid according to their physico-chemical properties. In addition, they were found in BrS patients with no other TRPM4 variants (and no SCN5A variants), a situation resulting in a simpler correlation between phenotype and genotype.Expression of TRPM4 VariantsTRPM4 current detection in the whole-cell configuration. Wild type (WT) TRPM4 and all mutantsFigure 1. ECG of patient 9 with Brugada features. (A) Unchallenged and (B) ajmaline-challenged ECG of patient 9 showing a transition from Brugada type 2 to type 1. Note the 58-49-1 supplier characteristic ST segment elevation in V1, V2 and V3. doi:10.1371/journal.pone.0054131.gexhibited a characteristic outward rectifying current when recorded in the whole-cell configuration (figure 3 A). A significant decrease in current density was detected for p.Pro779Arg and p.Lys914X transfected cells, in comparison to WT transfected cells (figure 3 B). The p.Lys914X transfected cells exhibited a current density similar to non-transfected HEK-293 cells, indicating that the mutant did not induce additional current. Single channel conductance. In HEK-293 cells stably expressing wild type TRPM4, a classical TRPM4 single current was detected in the inside-out configuration (figure 4 A) with a linear current-voltage relationship, providing a single channel conductance of 21.160.6 pS (n = 9) in accordance with previous reports [18,20]. Inside-out patches from p.Lys914X mutant transfected cells did not exhibit any detectable current (n = 20) (figure 4 B). Thus, this mutant was not further investigated. All other mutants provided detectable currents with a current-voltage relationship satisfactorily fitted to a linear regression (figure 4 B) providing single channel conductances g similar to WT (figure 4 C). On single channel 3PO traces provided for WT and each mutant in figure 4, channel activity was higher at Vm = +40 mV than at 240 mV, indicating a channel sensitivity to voltage, a TRPM4 fingerprint. Anion to cation permeability ratio. The anion to cation permeability ratio was investigated in inside-out patches. Reducing internal NaCl concentration from 145 mM to 42 mM shifted the reversal potential (Vrev). A representative recording is provided for p.Leu1075Pro (figure 5 A). The reversal potential of 2562 mV (n = 4) for WT with 42 mM NaCl, corresponds to a permeability ratio PNa/PCl of 14.2 according to the Goldman Hodgkin Katz (GHK) equation. All mutants exhibited 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 23977191 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.E 3 missense mutations gave a Grantham score [19] of 89 or more. Grantham is a formula estimating difference between amino acid according to their physico-chemical properties. In addition, they were found in BrS patients with no other TRPM4 variants (and no SCN5A variants), a situation resulting in a simpler correlation between phenotype and genotype.Expression of TRPM4 VariantsTRPM4 current detection in the whole-cell configuration. Wild type (WT) TRPM4 and all mutantsFigure 1. ECG of patient 9 with Brugada features. (A) Unchallenged and (B) ajmaline-challenged ECG of patient 9 showing a transition from Brugada type 2 to type 1. Note the characteristic ST segment elevation in V1, V2 and V3. doi:10.1371/journal.pone.0054131.gexhibited a characteristic outward rectifying current when recorded in the whole-cell configuration (figure 3 A). A significant decrease in current density was detected for p.Pro779Arg and p.Lys914X transfected cells, in comparison to WT transfected cells (figure 3 B). The p.Lys914X transfected cells exhibited a current density similar to non-transfected HEK-293 cells, indicating that the mutant did not induce additional current. Single channel conductance. In HEK-293 cells stably expressing wild type TRPM4, a classical TRPM4 single current was detected in the inside-out configuration (figure 4 A) with a linear current-voltage relationship, providing a single channel conductance of 21.160.6 pS (n = 9) in accordance with previous reports [18,20]. Inside-out patches from p.Lys914X mutant transfected cells did not exhibit any detectable current (n = 20) (figure 4 B). Thus, this mutant was not further investigated. All other mutants provided detectable currents with a current-voltage relationship satisfactorily fitted to a linear regression (figure 4 B) providing single channel conductances g similar to WT (figure 4 C). On single channel traces provided for WT and each mutant in figure 4, channel activity was higher at Vm = +40 mV than at 240 mV, indicating a channel sensitivity to voltage, a TRPM4 fingerprint. Anion to cation permeability ratio. The anion to cation permeability ratio was investigated in inside-out patches. Reducing internal NaCl concentration from 145 mM to 42 mM shifted the reversal potential (Vrev). A representative recording is provided for p.Leu1075Pro (figure 5 A). The reversal potential of 2562 mV (n = 4) for WT with 42 mM NaCl, corresponds to a permeability ratio PNa/PCl of 14.2 according to the Goldman Hodgkin Katz (GHK) equation. All mutants exhibited 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 23977191 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.