Aberrant Deactivation-Induced Gain of Function in TRPM4 Mutant Is Associated with Human Cardiac Conduction Block

Article

Aberrant Deactivation-Induced Gain of Function in TRPM4 Mutant Is Associated with Human Cardiac Conduction Block Graphical Abstract TRPM4wt

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Wenying Xian, Xin Hui, Qinghai Tian, ..., Veit Flockerzi, Sandra Ruppenthal, Peter Lipp

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Mutation TRPM4A432T is associated with human cardiac disease TRPM4A432T mutation displays 4-fold slower calciumdependent denactivation

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Rational mutagenesis reveals the bulkiness of the amino acid as a key contributor

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Molecular modeling suggests a compaction of channel subdomain in TRPMA432T

Xian et al., 2018, Cell Reports 24, 724–731 July 17, 2018 ª 2018 The Author(s). https://doi.org/10.1016/j.celrep.2018.06.034

A mutation in TRPM4 (A432T) is linked to life-threatening cardiac conduction disturbances in patients. Using a combination of biophysical techniques, Xian et al. demonstrate that calciumdependent deactivation between heartbeats is aberrant and highlights the etiology of human cardiac channelopathies. These findings offer putative new pharmacological targets for disease management in human patients.

Cell Reports

Article Aberrant Deactivation-Induced Gain of Function in TRPM4 Mutant Is Associated with Human Cardiac Conduction Block Wenying Xian,1 Xin Hui,1 Qinghai Tian,1 Hongmei Wang,4 Alessandra Moretti,2,3 Karl-Ludwig Laugwitz,2,3 Veit Flockerzi,4 Sandra Ruppenthal,1 and Peter Lipp1,5,* 1Molecular

Cell Biology, Centre for Molecular Signaling (PZMS), Medical Faculty, Saarland University, 66421 Homburg, Germany €nchen, 81675 Mu €nchen, Germany of Medicine I (Cardiology and Angiology), Klinikum rechts der Isar, Technische Universita¨t Mu 3DZHK (German Centre for Cardiovascular Research), partner site Munich Heart Alliance, Munich, Germany 4Experimental and Clinical Pharmacology and Toxicology, Centre for Molecular Signaling (PZMS), Medical Faculty, Saarland University, 66421 Homburg, Germany 5Lead Contact *Correspondence: [email protected] https://doi.org/10.1016/j.celrep.2018.06.034 2Department

SUMMARY

A gain-of-function mutation in the Ca2+-activated transient receptor potential melastatin member 4 (TRPM4A432T) is linked to life-threatening cardiac conduction disturbance, but the underlying mechanism is unclear. For deeper insights, we used photolysis of caged Ca2+, quantitative Ca2+, and electrophysiological measurements. TRPM4A432T’s 2-fold larger membrane current was associated with 50% decreased plasma membrane expression. Kinetic analysis unveiled 4-fold slower deactivation that was responsible for the augmented membrane current progressively rising during repetitive human cardiac action potentials. Rational mutagenesis of TRPM4 at position 432 revealed that the bulkiness of the amino acid was key to TRPM4A432T’s aberrant gating. Charged amino acids rendered the channel non-functional. The slow deactivation caused by an amino acid substitution at position 432 from alanine to the bulkier threonine represents a key contributor to the gain of function in TRPM4A432T. Thus, our results add a mechanism in the etiology of TRP channel-linked human cardiac channelopathies. INTRODUCTION Cardiac conduction block (CCB) manifests when the pacemaking signal initiated in the sinoatrial (SA) node is slowed down or even blocked during impulse propagation across the heart tissue (Liu et al., 2010) and can be life threatening (Michae¨lsson et al., 1995). Implantation of artificial pacemakers is still the main therapeutic option for a correct electrical conductance (Michae¨lsson et al., 1995; Vardas and Ovsyscher, 2002). A subset of CCB is caused by inherited mutations in genes coding for ion channels (Friedrich et al., 2014; Park and Fishman, 2011; Rossenbacker et al., 2004; Watanabe et al., 2008).

The transient receptor potential melastatin (TRPM) 4 channel is a Ca2+-activated nonselective channel permeable to Na+ and K+ (Amarouch et al., 2013; Launay et al., 2002). Its threedimensional (3D) structure was resolved recently, confirming a tetrameric protein complex (Autzen et al., 2018; Duan et al., 2018; Guo et al., 2017; Winkler et al., 2017). Mutations in TRPM4 have been linked to CCB (Daumy et al., 2016; Kruse et al., 2009; Liu et al., 2010; Stallmeyer et al., 2012) and Brugada syndrome (Liu et al., 2013). In three families with autosomaldominant isolated cardiac conduction disease (ICCD), three heterozygous missense mutations of the TRPM4 gene have been found in each family (R164W, A432T, and G844D), putatively increasing membrane currents through the same mechanism as proposed for TRPM4E7K (Liu et al., 2010). However, Syam et al. (2016) showed decreased plasma membrane expression and ionic current for TRPM4A432T. Up to now, gain or loss of function has been attributed solely to quantitative changes of the protein in the plasma membrane. As a Ca2+-activated channel, the established approach to study the electrophysiological properties of TRPM4 is to clamp the intracellular Ca2+ concentration through the pipette solution (Kruse et al., 2009; Liu et al., 2010). During dialysis, controlled changes in the intracellular Ca2+ concentration are challenging (Ullrich et al., 2005). A study with human TRPM5 showed that the temporal dynamics of intracellular Ca2+ may be important for channel activation (Prawitt et al., 2003). To provide controlled changes in Ca2+ and more physiological kinetics of Ca2+ changes, the methodology of caged Ca2+ compounds such as 2-nitro-4, 5-dimethoxyphenyl-EDTA (Kaplan and Ellis-Davies, 1988) or o-nitrophenyl-EGTA (NP-EGTA) (Ellis-Davies and Kaplan, 1994) might offer better experimental strategies for studying TRPM4’s properties. UV flash-mediated uncaging of such caged compounds is spatially homogeneous (Lipp et al., 1996). The combination of Ca2+ uncaging and electrophysiological measurement has been widely used (Go´mez et al., 2002; Lipp et al., 1996; Lipp and Niggli, 1994, 1998). In the present study, we used a UV flash approach to study the kinetic properties of TRPM4 channels and several mutant proteins. Our results indicate that alanine432 is a critical amino

724 Cell Reports 24, 724–731, July 17, 2018 ª 2018 The Author(s). This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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Figure 1. TRPM4A432T Mutation Displays Altered Kinetic Properties in Dynamic Ca2+ Assay (A and B) TRPM4 currents were activated by UV flashes in HEK293 cells transiently expressing TRPM4WT (A) and TRPM4A432T (B). (C) Typical current/Ca2+ relationships for TRPM4WT and TRPM4A432T (left) and statistical summary (right). (D) Statistical analysis of the maximal membrane current density normalized to the peak Ca2+ concentration. (E and F) Ca2+-dependent activation (E) and deactivation (F) of TRPM4 variants. Left: the time courses of the Ca2+ concentration (top) and corresponding normalized membrane currents (bottom). (E) Upper right: ‘‘raw’’ membrane currents; lower right: statistical summary of the activation time constants. (F) Upper right: decay time constants of the underlying Ca2+ transients; lower right: statistical summary of the deactivation time constants. (G–I) Expression analysis of TRPM4 variants (WT in black and A432T mutant in red). Arrows in (G) highlight the highly (closed arrowhead) and core glycosylated (open arrowhead) TRPM4 variant. Typical western blots of whole-cell lysates (G, left) and the statistical analysis (G, right). Surface expression exemplified (H, left) and statistical summary (H, right). The normalized ratios of highly glycosylated and core glycosylated TRPM4 proteins are depicted in (I). Bar graphs and error bars in this figure depict means values ±SEM. Experiments in (A)–(F) were performed at a holding potential of +80 mV.

acid determining a multitude of TRPM4’s properties. Most important, we also provide the first experimental evidence that an aberrant kinetic in the Ca2+-activated channel TRPM4 might be a novel etiology of human cardiac conduction disorders. RESULTS Gain of Function in TRPM4A432T Is Associated with Aberrant Deactivation and Lower Plasma Membrane Expression We used fast photolytic uncaging of caged Ca2+ to study TRPM4’s current amplitude, KD,Ca, and activation and deactivation characteristics (Figures S1 and S2).

In HEK293 cells expressing wild-type TRPM4 (TRPM4WT) and TRPM4A432T proteins, current recordings during trains of UV flashes indicated a substantially increased relative membrane current (Figures 1A, 1B, and 1D; 190.7 ± 21.82 versus 68.03 ± 12.70 pA/pF/mM for TRPM4A432T and TRPM4WT, respectively) with an unchanged apparent KD,Ca (Figure 1C). This gain of function was associated with 50% decreased cell surface expression (Figure 1H), while whole-cell expression was comparable (Figure 1G). Our kinetic analysis revealed aberrant deactivation kinetics that were slowed down by a factor of almost 4 (Figure 1F) with unchanged current activation (Figure 1E). Further investigations of the protein expression revealed the characteristic highly and core glycosylated proteins in whole-cell lysates Cell Reports 24, 724–731, July 17, 2018 725

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(Figure 1G, closed and open arrowheads, respectively) (Syam et al., 2014). Their relative expression levels were substantially changed for the TRPM4A432T mutant in that the occurrence of the highly glycosylated protein was only 20% of that of the wild-type (WT) protein (Figure 1I). In a series of experiments, we confirmed increased membrane currents and membrane expression of another TRPM4 gain-offunction mutation, TRPM4E7K (Kruse et al., 2009). This mutant did not display any altered kinetics (Figure S3). TRPM4A432T Generates Excessive Membrane Currents during Human Cardiac Action Potentials To gain additional insights into the disease contributions of the A432T mutation, especially also in comparison with the well characterized E7K mutation, we carried out an action potential playback experiment. To simultaneously mimic increases in Ca2+ that accompany cardiac action potentials, we uncaged Ca2+ (Figure 2B) immediately after the onset of the action potential (Figures 2A–2C, vertical dashed lines). Expression of the WT variant and the E7K mutant of TRPM4 displayed small and comparable membrane currents (Figures 2C–2F, black and cyan trace and symbols). In contrast, the TRPM4A432T variant depicted substantially deformed and progressively increasing membrane currents (Figures 2C–2F, red trace and symbols). Thus, although both E7K and A432T are gain-of-function mutations, they displayed very different current contributions during cardiac action potentials. These data suggest that TRPM4 channel mutants with altered kinetic properties might contribute to the occurrence of human cardiac arrhythmias. TRPM4’s Aberrant Deactivation Kinetics Are Independent of Glycosylation, cPKC-Dependent Phosphorylation, and Ca2+-Dependent Changes in PIP2 Levels Next, we wondered whether the overall reduced protein glycosylation per se might be linked to the channel’s altered deactivation. Application of tunicamycin (an antibiotic to inhibit 726 Cell Reports 24, 724–731, July 17, 2018

Vertical dashed lines indicate time points for the UV flashes applied to the NP-EGTA loaded cells. (A) Action potentials from a typical ventricular hiPS-CM. (B) Representative trace of the intracellular Ca2+ concentration. (C) Typical membrane currents resulting from the command voltage profile shown in (A). (D and E) Statistical analysis of a population of HEK293 cells. Membrane currents were probed at the given time points plotted against the time point. Color-coding corresponds to the colors used in (C). Note the linear scale in (D). For (E) we replotted the values on a semi-logarithmic scale to allow appreciation of the lower current components. (F) Replots of those parts of the recordings in (A)– (C) highlighted by the gray background at a higher magnification. Data points and error bars in (D) and (E) depict means values ± SEM, respectively.

protein glycosylation; 10 mg/mL for 24 hr) reduced TRPM4WT’s highly glycosylated form by more than 50% (Figure S4A) without altering its deactivation kinetics (Figure S4B). In addition to existing protein kinase C (PKC) phosphorylation sites in TRPM4WT (Nilius et al., 2005), the A432T mutation adds another threonine available for phosphorylation, and we asked whether Ca2+-dependent cPKC-mediated TRPM4 phosphorylation might be affecting deactivation kinetics. Application of the cPKC inhibitor Go¨6983 (1 mM) (Hui et al., 2014b) did not alter deactivation kinetics of both TRPM4WT and TRPM4A432T proteins (Figure S4C). TRPM4 channel behavior is modulated by plasma membrane PIP2 (Nilius et al., 2006; Zhang et al., 2005). U73122 (10 mM) is a potent inhibitor of phospholipase Cs (PLCs) (Hui et al., 2014a, 2014b), but its application did not alter deactivation of TRPM4WT (Figure S4D). Despite the reported increased TRPM4WT current density (Leitner et al., 2016), the activation was also unchanged (Figure S4E). These data indicated that neither an altered glycosylation nor cPKC-induced phosphorylation or plasma membrane PIP2 levels contributed to TRPM4A432T’s altered deactivation. The Bulkiness of the Amino Acid at Position 432 Contributes to TRPM4’s Channel Gating Behavior The amino acid at position 432 appears to represent a key position determining essential TRPM4 properties. We therefore systematically replaced alanine432 with other amino acids. In TRPM4WT, alanine432 displays a non-polar, small side chain. We modulated the bulkiness of this side chain by replacing alanine with the smallest possible non-polar amino acid glycine or with a slightly bulkier side chain with valine (Figure 3A). As shown in Figure 3, increasing the size of the residue (TRPM4A432V) resulted in a substantial gain of function (Figure 3B, middle, and Figure 3E in green). The deactivation time constant was increased more than 2-fold compared with that of the WT protein (Figures 3C and 3G in green; 7.262 ± 0.5224 s [n = 10] versus 3.187 ± 0.3498 s [n = 10], respectively)

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Figure 3. Bulkiness of the Amino Acid at Position 432 Is Critical for TRPM4’s Properties (A) Structure and polarity of TRPM4 variants. Color coding for this figure indicated by the color of the rectangles. (B) UV flash photolysis-induced Ca2+ jumps (left), the resulting activation time course of the induced membrane current (middle), and the normalized current upstrokes (right). (C) Decay period of the appropriate Ca2+ transients (left) and normalized current deactivation (right). (D–G) Statistical evaluation of KD,Ca (D), current amplitude (E), activation (F), and deactivation (G) time constant. (H and I) Expression analysis of three TRPM4 variants on the global level (H) and the plasma membrane level (I). The analysis of the ratio of highly glycosylated and core glycosylated TRPM4 proteins are summarized (H, bottom). Bar graphs and error bars in this figure depict means values ± SEM.

and was associated with faster activation (Figure 3B, right, and Figure 3F in green). Although its membrane expression was not significantly different from TRPM4WT (Figure 3I, green), the abundance of the highly glycosylated protein was reduced by more than 50% (Figure 3H, green, top and bottom). Decreasing the bulkiness of the residue (substitution by glycine) showed opposite effects compared with the valine substitution for almost all the parameters tested. Compared with the WT channel, TRPM4A432G can best be characterized as a loss-of-function mutation (Figures 3B and 3E in blue), with an only slightly increased apparent KD,Ca (Figure 3D, blue). The activation as well as deactivation properties were unchanged compared with TRPM4WT but different from those of the TRPM4A432V protein (Figures 3F and 3G in blue). Total as well as surface abundance and protein glycosylation were unaffected in the TRPM4A432G mutation (Figures 3Hand 3I in blue).

These data supported our notion that it is the bulkiness of the amino acid at site 432 that modulates TRPM4’s gating kinetics. To further investigate the effects of substituting alanine432 with a charged amino acid, we expressed TRPM4A432D (negative charge) and TRPM4A432K (positive charge) (Figure 4A). Both lacked substantial Ca2+-activated currents (Figures 4B and 4C) despite similar global but slightly reduced plasma membrane expression (Figures 4E and 4F). Expression of the highly glycosylated protein was greatly reduced for both variants (Figure 4E). The resulting current/Ca2+ relationships for TRPM4A432D and TRPM4A432K resembled those found in nontransfected cells rather than those from cells expressing TRPM4WT (Figure 4D). From these data we concluded that both proteins (i.e., TRPM4A432D and TRPM4A432K) did not generate detectable levels of Ca2+-activated membrane currents. Cell Reports 24, 724–731, July 17, 2018 727

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Figure 4. Charged Amino Acid at Position 432 Renders TRPM4 Proteins Non-functional (A) Structure and charges of TRPM4 variants. Color coding for this figure indicated by the color of the rectangles. (B) Left: UV flash photolysis-induced Ca2+ jumps and the resulting activation time course of the induced membrane current (right). (C) Maximal current density induced by the Ca2+ jumps in a train of UV flashes. (D) Typical membrane current/Ca2+ relationship for the TRPM4 variants and non-transfected HEK293 cells (gray). (E and F) Expression analysis of the three TRPM4 variants on the global level (E) and the plasma membrane level (F). The analysis of the ratio of highly glycosylated and core glycosylated TRPM4 proteins are summarized in (E, bottom). Bar graphs and error bars in this figure depict means values ± SEM.

Mutations at Position 432 Change the Compactness of TRPM4’s Homology Region 3 Recent reports on the structure of the TRPM4 protein described four homology regions (MHRs) within the TRPM4 protein, and position 432 resides in MHR3, which bridges between the MHR1/2 and MHR4 domains (Autzen et al., 2018; Winkler et al., 2017). The proposed structure of TRPM4 provides a suitable template for structural modeling of putative steric consequences of point mutations at position 432 (Figure 5). We performed simulations and refinement of TRPM4’s structure and compared the MHR3 domains among all variants. Alanine432 forms hydrophobic interactions with the surrounding residues V401, P438, and F440 to maintain a functional structure (Figure 5A). In A432T, the threonine might interact with more residues (V401, R437, P438, F440, V441, and L444), thereby strengthening the interaction within MHR3 domain and yielding a more compact structure (Figure 5B). A similar situation results from A432V (Figure 5D), whereas A432G without side chain is more flexible, which may regionally disrupt the a-helix in the MHR3 domain to form a random coil (Figure 5C). Because of the bulkiness of aspartate and lysine, in A432D and A432K, 728 Cell Reports 24, 724–731, July 17, 2018

repulsion forces from interactions with residues L397, V401, F414, S428, L429, D431, L433, R437, P438, F440, V441, and L444 might strongly affect surrounding helix movement, loosening and destabilizing the MHR3 domain (Figures 5E and 5F). DISCUSSION TRPM4 is among the most important cardiac TRP channels for which direct links to human cardiac arrhythmias have been described (Daumy et al., 2016; Kruse et al., 2009; Liu et al., 2010; Stallmeyer et al., 2012; Syam et al., 2016; Brink and Torrington, 1977; Stephan, 1978). We have set up assays closely resembling the rapid and dynamic increases of intracellular Ca2+ in cardiac myocytes to kinetically characterize TRPM4mediated membrane currents while avoiding exposure of the cell to extended periods of excessively high Ca2+ concentrations. Single Ca2+ transients elicited by an individual UV flash permitted the analysis of activation and deactivation kinetics, and a train of Ca2+ jumps resulted in a wider range of Ca2+ concentrations from which we derived quantitative current/ Ca2+ relationships (Figure S1). For TRPM4WT, our data compare

Figure 5. Model of TRPM4’s MHR3 Domain (A) The alanine432 of the TRPM homology region (MHR) 3 forms hydrophobic interactions with adjacent V401, P438, and F440 residues (inset). (B) In the A432T variant, the threonine might interact with additional residues (V401, R437, P438, F440, V441, and L444; inset), strengthening the interactions within MHR3. (C) The glycine of A432G is more flexible, forming a random coil. (D) Like the threonine of A432T, the valine residue of A432V favors more hydrophobic interactions, yielding a more compact MHR3 configuration in comparison with the wild-type A432. (E and F) Replacement of A432 by negatively (E) and positively (F) charged amino acid residues may promote interactions with L397, V401, F414, R437, P438, F440, V441, and L444, which affect the surrounding helix movement, causing repulsion of the helices and destabilizing the MHR3 domain. The structure is based on the coordinates of human TRPM4 (Winkler et al., 2017) (PDB: 5WP6).

well with data published earlier (Mathar et al., 2014; Ullrich et al., 2005). Although our UV flash method did not produce stable Ca2+ plateaus following the uncaging event, this was uncritical for the determination of the apparent KD,Ca (Figure S2). The TRPM4A432T mutation has been associated with childhood and adult cardiac arrhythmias (Liu et al., 2010; Syam et al., 2016) and was described as both loss of function (Syam et al., 2016) and gain of function (Liu et al., 2010; Stallmeyer et al., 2012). The hypothesized underlying molecular mechanism involved was altered SUMOylation, as suggested for the E7K mutation. In contrast, our experimental approach revealed a multitude of changes far beyond changes of protein abundance (Figure 1). Although the apparent KD,Ca and the activation time constant were unchanged, all other parameters were altered substantially (Figure 1). The reason for the augmented current density (Figure 1D) is currently unclear but might be found in an altered single-channel gating because of reduced membrane expression (Figure 1H). This will need further experimental attention. Most surprising, our kinetic analysis revealed a novel alteration of the TRPM4A432T mutation. Its deactivation was slowed down substantially, a so far unrecognized etiology in cardiac TRPM4-related channelopathies. Noteworthy, this deactivation is kinetically distinct from the inactivation that occurs during constant Ca2+ concentration and is about an order of magnitude faster (Nilius et al., 2005). To estimate the resulting membrane currents during cardiac action potentials, our striking findings suggest that two important and synergistic requirements need to coincide for TRPM4A432T to produce exaggerated membrane currents: repetitive stimulations and elevated Ca2+ concentrations. Both result in the generation of gradually increasing depolarizing membrane currents, a direct consequence of the slowed-down deactivation kinetics. In cardiac myocytes, all maneuvers that will increase Ca2+ concentration and/or the firing rate of the cell might unavoidably result in a vicious circle driven by

exaggerated production of depolarizing TRPM4 membrane currents. The resulting current will substantially distort the shape of the cardiac action potential and may alter impulse propagation. We are aware that this methodology has drawbacks compared with the ‘‘real’’ cardiac environment. One of the most prominent is the obvious absence of other contributing current components that shape the cardiac action potential and functionally interact with the TRPM4 current. In addition, the lipid composition of the plasma membrane is different between HEK293 cells and cardiac myocyte, and, for example, PIP2 has a substantial impact on TRPM4’s behavior (Nilius et al., 2006). Nevertheless, we are confident that the lack of pharmacological interventions, the use of human action potentials, the ability to experimentally manipulate the intracellular Ca2+ concentration, and the shortage of cardiac myocytes from affected patients warrant this experimental approach. We identified the bulkiness of threonine as a potent contributor to the altered properties of TRPM4A432T (Figures 3 and 4). On the basis of recent electron cryomicroscopy structures of human TRPM4 (Winkler et al., 2017), we propose that the amino acid at position 432 modulates the compactness of TRPM4’s MHR3 domain (Figure 5). The structure of the Ca2+ occupied TRPM4 protein suggests that the cytoplasmic part of TRPM4 may be involved in the regulation of TRPM4’s gating behavior (Autzen et al., 2018). The TRP helix of the C terminus forms direct interactions with the MHR domain of the N terminus, suggesting a mechanism by which MHR interacts with the TRP helix to modulate TRPM4’s gating (Winkler et al., 2017). In conclusion, our study revealed an important mechanism by which Ca2+-activated TRPM4’s aberrant deactivation might substantially contribute membrane currents to the cardiac action potential, eventually distort its shape, and result in aberrant impulse propagation and distribution on the tissue level. Such etiologies could be revealed only by considering and mimicking both dynamic membrane potential changes and, in the case of a Cell Reports 24, 724–731, July 17, 2018 729

Ca2+-activated membrane current, also the ‘‘systolic’’ Ca2+ dynamics of a cardiac myocyte. It is important to also emphasize the contribution of the diastolic Ca2+ concentration. Elevations of diastolic Ca2+, similar to those described in many cardiac diseases (Bers, 2002; Jung et al., 2012; Neef et al., 2010; Paavola et al., 2007), will ‘‘prime’’ mutant Ca2+-dependent membrane channels, such as TRPM4A432T, for the generation of additional, diastolic membrane currents. Action potentials and/or diastolic membrane potentials altered in such a way represent an ideal substrate for the occurrence of arrhythmic cardiac events such as life-threatening conduction blocks. EXPERIMENTAL PROCEDURES Electrophysiology Whole-cell membrane currents were measured in the whole-cell patch clamp with an EPC-10 amplifier (HEKA Electronic, Germany). Patch pipettes had a resistance of 3–5 MU. The free Ca2+ concentration of the pipette solution was determined in independent experiments in small aliquots of the solution by Fura-2 in vitro. Human induced pluripotent stem cell-derived cardiac myocytes (hiPS-CMs) were differentiated and dissociated as described recently (Chen et al., 2016). For the generation of hiPSCs, recruitment and consenting procedures were executed under the institutional review board-approved protocols of Klinikum rechts der Isar and written informed consent was obtained. Seventy-two hr after seeding, spontaneous action potentials were recorded from hiPS-CMs. For playback experiments in HEK293 cells, typical ventricular-like action potential waveforms were selected as the command voltage template in voltage-clamp. This protocol also included triggering of UV flashes and simultaneous Ca2+ recording as described below. 2+

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Photolytic Release of Ca and Measurement of [Ca ]i Ca2+ uncaging was achieved by brief high-intensity flashes from a UV flash lamp (RAPP OptoElectronic, Germany) coupled into the microscope by reflection from a 420 nm long-pass dichroic mirror. Fluo-4FF was excited at 480 ± 5 nm with a monochromator (PolyChrome V, TILL, Photonics, Germany), and the emission was collected through a 515 nm long-pass filter onto an avalange photodiode-based epifluorescence hardware (TILL, Photonics, Germany). The Ca2+ concentration was calculated as described previously (Cheng et al., 1993). The procedure for constructing current/Ca2+ relationships is explained in detail in Figure S1. Determination of the Activation and Deactivation Time Constants Figure S1A details the two time periods used for determination of the activation and deactivation time constants. Fittings were calculated with equations in Prism software (see Supplemental Experimental Procedures). The determination of the membrane current’s deactivation time constant is restricted by the decay kinetics of the underlying Ca2+ transient. Site-Directed Mutagenesis and MHR3 Modeling The human TRPM4 cDNA was used as template for site directed mutagenesis. The mutant cDNA constructs were sequenced on both strands before use. Figure 5 was prepared using PyMOL Molecular Graphics System (version 1.5.0.4, Schro¨dinger, New York, NY, USA) by Coot30 (Emsley et al., 2010), on the basis of the coordinates of human TRPM4 (Winkler et al., 2017) (PDB: 5WP6). Statistical Analysis Results are presented as mean ± SEM. All resulting data were initially tested for normal distribution in Prism 7.0 (GraphPad Software, USA) using the D’Agostino and Pearson normality test. All data in this report passed the normality test unless otherwise stated. In cases in which two datasets were to be analyzed, the unpaired two-tailed Student’s t test was used. In cases with more than two groups, we used an ordinary one-way ANOVA followed by a column-against-each-column posttest. Significance is characterized by

730 Cell Reports 24, 724–731, July 17, 2018

particular p values: *p < 0.05, **p < 0.01, and ***p < 0.005. ‘‘n’’ numbers are given as the number of investigated cells considering that the data were collected from at least two independent set of experiments. For western blot analysis, the ‘‘n’’ number represents the number of transfections. SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures and five figures and can be found with this article online at https://doi.org/ 10.1016/j.celrep.2018.06.034. ACKNOWLEDGMENTS Part of this work was funded by the TRR SFB 152 (Deutsche Forschungsgemeinschaft [DFG]) to W.X., A.M., K.-L.L., V.F., and P.L. Q.T. acknowledges the support of intramural funding from the medical faculty (HOMFORexcellent). H.W. is a recipient of an Alexander von Humboldt research scholarship in the Flockerzi lab. AUTHOR CONTRIBUTIONS W.X., X.H., S.R., and A.M. performed the molecular laboratory work. W.X., Q.T., S.R., and X.H. designed and performed experiments. P.L., W.X., and V.F. designed the study. W.X. and P.L. analyzed data. H.W. and V.F. generated the MHR3 model. W.X., X.H., Q.T., H.W., A.M., K.-L.L., V.F., S.R., and P.L. contributed text and/or figure panels to the manuscript. P.L. and W.X. wrote the manuscript. All authors gave final approval for publication. DECLARATION OF INTERESTS The authors declare no competing interest. Received: November 7, 2017 Revised: February 27, 2018 Accepted: June 7, 2018 Published: July 17, 2018 REFERENCES Amarouch, M.-Y., Syam, N., and Abriel, H. (2013). Biochemical, singlechannel, whole-cell patch clamp, and pharmacological analyses of endogenous TRPM4 channels in HEK293 cells. Neurosci. Lett. 541, 105–110. Autzen, H.E., Myasnikov, A.G., Campbell, M.G., Asarnow, D., Julius, D., and Cheng, Y. (2018). Structure of the human TRPM4 ion channel in a lipid nanodisc. Science 359, 228–232. Bers, D.M. (2002). Cardiac excitation-contraction coupling. Nature 415, 198–205. Brink, A.J., and Torrington, M. (1977). Progressive familial heart block—two types. S. Afr. Med. J. 52, 53–59. Chen, Z., Xian, W., Bellin, M., Dorn, T., Tian, Q., Goedel, A., Dreizehnter, L., Schneider, C.M., Ward-van Oostwaard, D., Ng, J.K.M., et al. (2016). Subtype-specific promoter-driven action potential imaging for precise disease modelling and drug testing in hiPSC-derived cardiomyocytes. Eur. Heart J. 38, 292–301. Cheng, H., Lederer, W.J., and Cannell, M.B. (1993). Calcium sparks: elementary events underlying excitation-contraction coupling in heart muscle. Science 262, 740–744. Daumy, X., Amarouch, M.-Y., Lindenbaum, P., Bonnaud, S., Charpentier, E., Bianchi, B., Nafzger, S., Baron, E., Fouchard, S., Thollet, A., et al. (2016). Targeted resequencing identifies TRPM4 as a major gene predisposing to progressive familial heart block type I. Int. J. Cardiol. 207, 349–358. Duan, J., Li, Z., Li, J., Santa-Cruz, A., Sanchez-Martinez, S., Zhang, J., and Clapham, D.E. (2018). Structure of full-length human TRPM4. Proc. Natl. Acad. Sci. U S A 115, 2377–2382.

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