Variants of Transient Receptor Potential Melastatin Member 4 in Childhood Atrioventricular Block

Abstract

Background: Transient receptor potential melastatin member 4 (TRPM4) is a nonselective cation channel. TRPM4 mutations have been linked to cardiac conduction disease and Brugada syndrome. The mechanisms underlying TRPM4-dependent conduction slowing are not fully understood. The aim of this study was to characterize TRPM4 genetic variants found in patients with congenital or childhood atrioventricular block.

Methods and results: Ninety-one patients with congenital or childhood atrioventricular block were screened for candidate genes. Five rare TRPM4 genetic variants were identified and investigated. The variants were expressed heterologously in HEK293 cells. Two of the variants, A432T and A432T/G582S, showed decreased expression of the protein at the cell membrane; inversely, the G582S variant showed increased expression. Further functional characterization of these variants using whole-cell patch-clamp configuration showed a loss of function and a gain of function, respectively. We hypothesized that the observed decrease in expression was caused by a folding and trafficking defect. This was supported by the observation that incubation of these variants at lower temperature partially rescued their expression and function. Previous studies have suggested that altered SUMOylation of TRPM4 may cause a gain of function; however, we did not find any evidence that supports SUMOylation as being directly involved for the gain-of-function variant.

Conclusions: This study underpins the role of TRPM4 in the cardiac conduction system. The loss-of-function variants A432T/G582S found in 2 unrelated patients with atrioventricular block are most likely caused by misfolding-dependent altered trafficking. The ability to rescue this variant with lower temperature may provide a novel use of pharmacological chaperones in treatment strategies.

Keywords: atrioventricular block; mutations; temperature‐dependent rescue; transient receptor potential melastatin member 4.

Figure 1

Families with AVB. A, Pedigrees of families with members harboring TRPM4 variants. B, Illustration of the TRPM4 channel showing the location of the 5 congenital atrioventricular block variants described in this study. The dashed box highlights the presence of 2 variants in the same patient. AVB indicates atrioventricular block.

Figure 2

Human, mouse, and rat TRPM4 sequence alignments and localization of variants described in this study.

Figure 3

ECGs of patients harboring TRPM4 variants. A, ECG of a girl aged 14 years showing severe bradycardia caused by complete atrioventricular block, with narrow QRS complexes (heart rate 37 beats/min, QRS complex 69 ms). B, ECG in a man aged 45 years showing atrioventricular block type 1 (PR interval 240 ms) associated with an incomplete left bundle branch block (RS pattern in right precordial leads, RS pattern in left precordial leads, QRS complex 110 ms, QRS axis −15°). C, ECG in a girl aged 7 years showing atrioventricular block type 1 (PR interval 280 ms) associated with incomplete right bundle branch block (RSR′ pattern in right precordial leads, QRS complex 94 ms, QRS axis 135°). D, ECG in a man aged 47 years showing atrioventricular block type 1 (PR interval 220 ms) associated with incomplete left bundle branch block (RS pattern in right precordial leads, RS pattern in left precordial leads, QRS complex 100 ms, QRS axis −35°). E, ECG in a girl aged 7 years showing bradycardia caused by complete atrioventricular block with narrow QRS complex (heart rate 40 beats/min, QRS complex 80 ms). F, Holter ECG in a boy aged 6 months showing severe bradycardia caused by complete atrioventricular block with narrow QRS complexes (heart rate 55 beats/min, QRS complex 65 ms). G, Holter ECG in a boy aged 2 years showing severe bradycardia caused by 2/1 atrioventricular block with narrow QRS complexes (heart rate 40 beats/min, QRS 80 ms).

Figure 4

Expression of TRPM4 WT and atrioventricular block variants. A, Western blots showing the expression of TRPM4 at the total (left panel) and surface levels (right panel), with black and white arrowheads representing fully glycosylated and core‐glycosylated forms of TRPM4, respectively. B, Quantification of the Western blots is shown as relative intensity of protein bands for both fully and core‐glycosylated forms of TRPM4 in each fraction. Quantification done on 4 different Western blots. *P<0.05, **P<0.01, ***P<0.001. WT indicates wild type.

Figure 5

Whole‐cell patch‐clamp recording of WT and TRPM4 variants. A, Time course recording of TRPM4 current. B, Individual current traces of each WT and TRPM4 variants recorded as plateau phases. C, Quantification of current density of WT and atrioventricular block variants. *P<0.05, **P<0.01, ***P<0.001 (WT, n=20; D198G, n=6; A432T, n=6; A432T/G582S, n=5; G582S, n=6; T677I, n=5; V921I, n=5; and E7K, n=6). WT indicates wild type.

Figure 6

Role of SUMOylation. Immunoprecipitation with anti‐RanGAP1 and anti‐HA. A, Western blots on the upper panel show immunoprecipitation of RanGAP1 and TRPM4, (B) whereas the lower panels show their SUMOylation status. All samples were run on the same gel and were blotted multiple times on the same membrane with different antibodies. The lanes were rearranged for clarity, and each experiment was performed 3 times. C, Normalized current density of WT TRPM4 coexpressed with either Ubc9 or the catalytically inactive mutant Ubc9*. *P<0.05 (WT, n=8; Ubc9, n=5; and Ubc9*, n=5). WT indicates wild type.

Figure 7

Rescue experiments of WT and TRPM4 variants. A, Western blot and quantification showing expression of WT and TRPM4 variants at the total and surface levels at 37°C and 28°C. Quantification was done on 3 different Western blots. B, Current density of WT and A432T at 37°C and 28°C recorded as the plateau phase. *P<0.05, **P<0.01 (WT at 28°C, n=4; WT at 37°C, n=6; A432T at 28°C, n=6; and A432T at 37°C, n=6). Temp indicates temperature; WT, wild type.

Figure 8

Ubiquitylation of TRPM4. A, GST pull‐down experiment using GST‐tagged S5a (S), a subunit proteasome that recognizes ubiquitylated proteins, and GST alone (G) to pull down TRPM4 from HEK293 cells transiently transfected with WT TRPM4, A432T, and A432T/G582S individually. The input fraction is shown at left, and the pull‐down fraction is shown at right. B, Quantification of each fraction and comparison of WT to variant ratios between fractions. Quantification was done on 3 different Western blots. GST indicates glutathione‐S‐transferase; n.s., not significant; WT, wild type.