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. 2013;8(1):e54131.
doi: 10.1371/journal.pone.0054131. Epub 2013 Jan 30.

Molecular genetics and functional anomalies in a series of 248 Brugada cases with 11 mutations in the TRPM4 channel

Hui Liu  1 , Stéphanie Chatel, Christophe Simard, Ninda Syam, Laurent Salle, Vincent Probst, Julie Morel, Gilles Millat, Michel Lopez, Hugues Abriel, Jean-Jacques Schott, Romain Guinamard, Patrice Bouvagnet
Affiliations

Molecular genetics and functional anomalies in a series of 248 Brugada cases with 11 mutations in the TRPM4 channel

Hui Liu et al. PLoS One. 2013.

Abstract

Brugada syndrome (BrS) is a condition defined by ST-segment alteration in right precordial leads and a risk of sudden death. Because BrS is often associated with right bundle branch block and the TRPM4 gene is involved in conduction blocks, we screened TRPM4 for anomalies in BrS cases. The DNA of 248 BrS cases with no SCN5A mutations were screened for TRPM4 mutations. Among this cohort, 20 patients had 11 TRPM4 mutations. Two mutations were previously associated with cardiac conduction blocks and 9 were new mutations (5 absent from ~14'000 control alleles and 4 statistically more prevalent in this BrS cohort than in control alleles). In addition to Brugada, three patients had a bifascicular block and 2 had a complete right bundle branch block. Functional and biochemical studies of 4 selected mutants revealed that these mutations resulted in either a decreased expression (p.Pro779Arg and p.Lys914X) or an increased expression (p.Thr873Ile and p.Leu1075Pro) of TRPM4 channel. TRPM4 mutations account for about 6% of BrS. Consequences of these mutations are diverse on channel electrophysiological and cellular expression. Because of its effect on the resting membrane potential, reduction or increase of TRPM4 channel function may both reduce the availability of sodium channel and thus lead to BrS.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 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.
Figure 2
Figure 2. Electrophoregrams of 3 missense mutations and localization of TRPM4 mutations (A–C).
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).
Figure 3
Figure 3. Biophysical properties of WT and mutants TRPM4 channel in whole-cell configuration.
(A): Representative current tracings recorded in the whole cell conditions (ramp protocol under the traces) (B): Mean current density for WT and mutants estimated using the maximal current recorded during the ending step of 20 ms at Vm = +100 mV (see A) and reported to cell capacitance. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n.s.: not significant. Numbers in bars = number of experiments. Error bar: standard error of the mean.
Figure 4
Figure 4. Single channel currents.
Inside-out single channel currents for WT and mutants TRPM4. Representative recordings at Vm = +40 and −40 mV and mean current/voltage relationship of WT (A) and mutants (B). No significant currents were detected for K914X mutant. (C): Single channel conductance g of WT and mutants. Mean values for 5 to 9 experiments. ND: not determined.
Figure 5
Figure 5. Na+/Cl− permeability ratio and channels regulation.
(A): PNa/PCl permeability ratio was estimated by changing the 145 mM NaCl solution to a 42 mM NaCl solution to measure the shift of the current-voltage relationship. Voltage ramp protocol from Vm = −100 to +100 mV was applied as showed for L1075P. (B): Reversal potential (Vrev) was estimated for WT and mutants as showed in A. PNa/PCl was calculated according to the GHK equation. Similar results were obtained for mutants and WT. (C): Effect of [Ca2+]i on unitary channel activity was evaluated at Vm = +40 mV by reducing [Ca2+]i from 10−3 M to 10− 6 M. A representative trace is provided for WT. Magnification allows observing single-channel currents. Label “c” indicates the current level corresponding to the closed state of all channels. (D): Mean % of inhibition of channel activity with [Ca2+]i  = 10− 6 M compare to 10− 3 M for WT and mutants. No significant differences were detected. (E): Channel sensitivity to voltage was evaluated in the whole-cell configuration by estimating NPo in function of voltage for each mutant during ramp protocols (see proceedings description in the text) as showed for WT and P779R. (F): Mean voltage for half maximal activity (V1/2) estimated from traces as showed in E. P779R exhibited a significant increase in V1/2. ** = significantly different from WT (p<0.01). Numbers in bars = number of experiments.
Figure 6
Figure 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.
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Grants and funding

This work was supported by grants from Projet Hospitalier de Recherche Clinique 2008 and Foundation Renaud Febvre, les Etards, France to PB, and Swiss National Science Foundation TransCure and 310030 120707 grants to HA. The funders had no role in study design, decision to publish, or preparation of the manuscript.