Variants p.Thr873Ile and p.Leu1075Pro showed no alteration of whole-cell current, single channel properties, and TRPM4 channel regulation. Western blots showed an increasedTRPM4 Mutations in Brugada SyndromeFigure 6. Expression of TRPM4 channel in whole cell extracts and plasma membrane fraction. Several TRPM4 mutants show an alteration of protein expression at a total level as well as at the cell surface. (A) Whole cell lysates from HEK-293 cells transfected with TRPM4 constructs used as input, representing the total expression of TRPM4 protein. Quantification of the double bands, presumably fully and core glycosylated forms of TRPM4, black and white arrrows, respectively, is shown on the bottom panels. (B) The biotinylated fractions from the same transfection represent the amount of TRPM4 BIRB 796 expressed at the cell surface. Quantification of the double bands is shown on the bottom panels. n = 3. *p,0.05, **p,0.01, ***p,0.001. Error bar: standard error of the mean. doi:10.1371/journal.pone.0054131.gsurface expression although the full glycosylated expression of p.Thr873Ile did not reach a statistical threshold. The mechanisms linking TRPM4 functional alterations and ECG perturbations observed in BrS remain to be clarified. The main perturbation characteristic of the pathology is the STsegment elevation observed in ECGs. Two models have been proposed to account for the ST segment elevation in BrS: therepolarizing disorder and the depolarizing disorder hypothesis [21]. The repolarizing model is mainly based on the transmural voltage gradient caused by heterogeneity in action potential (AP) plateau among cells spanning the ventricular wall. Change in AP dome depends on modifications of currents activated during the early repolarization and plateau phases of the AP, mainly Ito, INaTRPM4 Mutations in Brugada Syndromeand ICa. TRPM4 may participate in AP shape by promoting the plateau. Due to its non-selective cationic selectivity, TRPM4 activation drives the membrane potential to 0 mV by conducting an outward repolarizing K+ current at positive voltages, but an inward depolarizing Na+ current at purchase PHA-739358 negative voltages. Because 1655472 TRPM4 is activated by internal Ca2+, it is more likely to activate during the plateau phase when internal Ca2+ increased and thus counteracts repolarizing K+ currents. Thereby, modifications of TRPM4 expression by mutations would change AP dome. This might explain the effect of mutants leading to increased expression but not reduced expression since TRPM4 is only weakly expressed in normal mammalian ventricle [15,16,22]. On the other hand, the depolarizing model depends more on conduction delay in the right ventricular outflow tract (RVOT) than differences in AP shape. RVOT perturbations are presented as a substrate site for ventricular tachyarrhythmias [23,24]. While TRPM4 is poorly expressed in mammalian ventricle, it is more expressed in nodal tissue [15,16,25]. Interestingly, the embryologic origin of RVOT is similar to those of atrioventricular regions [21] but different from those of ventricles. According to this, TRPM4 might be abundantly expressed in RVOT. It can be speculated that in analogy to the phenomenon of supernormal excitability and conduction [26], both a gain-of-function and loss-of-function of TRPM4 channels may lead to conduction slowing by reducing the availability of Nav1.5 sodium channels. A gain-of-function may depolarize the resting membrane potential and thus inactivate sodium channels, while a loss-o.Variants p.Thr873Ile and p.Leu1075Pro showed no alteration of whole-cell current, single channel properties, and TRPM4 channel regulation. Western blots showed an increasedTRPM4 Mutations in Brugada SyndromeFigure 6. Expression of TRPM4 channel in whole cell extracts and plasma membrane fraction. Several TRPM4 mutants show an alteration of protein expression at a total level as well as at the cell surface. (A) Whole cell lysates from HEK-293 cells transfected with TRPM4 constructs used as input, representing the total expression of TRPM4 protein. Quantification of the double bands, presumably fully and core glycosylated forms of TRPM4, black and white arrrows, respectively, is shown on the bottom panels. (B) The biotinylated fractions from the same transfection represent the amount of TRPM4 expressed at the cell surface. Quantification of the double bands is shown on the bottom panels. n = 3. *p,0.05, **p,0.01, ***p,0.001. Error bar: standard error of the mean. doi:10.1371/journal.pone.0054131.gsurface expression although the full glycosylated expression of p.Thr873Ile did not reach a statistical threshold. The mechanisms linking TRPM4 functional alterations and ECG perturbations observed in BrS remain to be clarified. The main perturbation characteristic of the pathology is the STsegment elevation observed in ECGs. Two models have been proposed to account for the ST segment elevation in BrS: therepolarizing disorder and the depolarizing disorder hypothesis [21]. The repolarizing model is mainly based on the transmural voltage gradient caused by heterogeneity in action potential (AP) plateau among cells spanning the ventricular wall. Change in AP dome depends on modifications of currents activated during the early repolarization and plateau phases of the AP, mainly Ito, INaTRPM4 Mutations in Brugada Syndromeand ICa. TRPM4 may participate in AP shape by promoting the plateau. Due to its non-selective cationic selectivity, TRPM4 activation drives the membrane potential to 0 mV by conducting an outward repolarizing K+ current at positive voltages, but an inward depolarizing Na+ current at negative voltages. Because 1655472 TRPM4 is activated by internal Ca2+, it is more likely to activate during the plateau phase when internal Ca2+ increased and thus counteracts repolarizing K+ currents. Thereby, modifications of TRPM4 expression by mutations would change AP dome. This might explain the effect of mutants leading to increased expression but not reduced expression since TRPM4 is only weakly expressed in normal mammalian ventricle [15,16,22]. On the other hand, the depolarizing model depends more on conduction delay in the right ventricular outflow tract (RVOT) than differences in AP shape. RVOT perturbations are presented as a substrate site for ventricular tachyarrhythmias [23,24]. While TRPM4 is poorly expressed in mammalian ventricle, it is more expressed in nodal tissue [15,16,25]. Interestingly, the embryologic origin of RVOT is similar to those of atrioventricular regions [21] but different from those of ventricles. According to this, TRPM4 might be abundantly expressed in RVOT. It can be speculated that in analogy to the phenomenon of supernormal excitability and conduction [26], both a gain-of-function and loss-of-function of TRPM4 channels may lead to conduction slowing by reducing the availability of Nav1.5 sodium channels. A gain-of-function may depolarize the resting membrane potential and thus inactivate sodium channels, while a loss-o.
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