Impaired endocytosis of the ion channel TRPM4 is associated with human progressive familial heart block type I

Progressive familial heart block type I (PFHBI) is a progressive cardiac bundle branch disease in the His-Purkinje system that exhibits autosomal-dominant inheritance. In 3 branches of a large South African Afrikaner pedigree with an autosomal-dominant form of PFHBI, we identified the mutation c.19G→A in the transient receptor potential cation channel, subfamily M, member 4 gene (TRPM4) at chromosomal locus 19q13.3. This mutation predicted the amino acid substitution p.E7K in the TRPM4 amino terminus. TRPM4 encodes a Ca²⁺-activated nonselective cation (CAN) channel that belongs to the transient receptor potential melastatin ion channel family. Quantitative analysis of TRPM4 mRNA content in human cardiac tissue showed the highest expression level in Purkinje fibers. Cellular expression studies showed that the c.19G→A missense mutation attenuated deSUMOylation of the TRPM4 channel. The resulting constitutive SUMOylation of the mutant TRPM4 channel impaired endocytosis and led to elevated TRPM4 channel density at the cell surface. Our data therefore revealed a gain-of-function mechanism underlying this type of familial heart block.

The electrical impulse of each heartbeat is propagated through the heart by direct cell-cell coupling of cardiac muscle cells and through the cardiac conduction system, composed of the sinoatrial node (the natural pacemaker [PM]), ill-defined atrial tracks, and the atrioventricular node, and thereafter the ventricular conduction system, formed by the bundle of His, the left and right bundle branches, and Purkinje fibers (1). Conduction block at the atrioventricular level or more distal (i.e., complete heart block [CHB]) is one of the most common indications for PM implantation, with 3 of 1,000 individuals in Europe receiving PMs (2). Because PM implantation has been shown to improve survival (3), conduction disease poses an important health problem. Causal loci have been mapped for single-gene diseases involving atrioventricular conduction in isolation or associated with cardiomyopathy or skeletal myopathy; for a number of diseases, the actual gene has been identified (4). One of these diseases is progressive familial heart block type I (PFHBI; also known as PFHBIB), an autosomal-dominantly inherited disease of the His-Purkinje system; a number of microsatellite markers (5) have mapped PFHBI to chromosome 19q13.3 (multipoint LOD score, 11.6, centering in a 10-cM interval around the kallikrein I [KLK1] locus; the highest 2-point LOD score for KLK1 is 6.49 with Θ of 0). A disease sharing phenotypic characteristics, described as isolated cardiac conduction disorder (ICCD) in a Lebanese kindred, has been mapped to the same chromosomal location (6). Furthermore, another progressive cardiac conduction disease sharing features with PFHBI is caused by mutations in a sodium channel, SCN5A, on chromosome 3 (7, 8). In PFHBI, disease progression is characterized initially by the occurrence of right bundle branch block (RBBB), followed by bifascicular block and finally CHB (9). Another conduction disease, PFHBII (also known as PFHB2), described at the same time as PFHBI, differs clinically: CHB occurrence is consistent with disease at an atrioventricular nodal level (9, 10), not with ventricular conduction disease.

In the PFHBI linkage study (5), the phenotype was based on ECG-defined RBBB, bifascicular block, or CHB with broad QRS complexes, or the presence of an implanted PM if clinical details indicating this intervention were unknown (Figure 1). Subsequent high-resolution mapping permitted identification of carriers and noncarriers based on the disease-associated haplotype (5), and the information has been used to counsel at-risk individuals about their carrier status.

Figure 1

Cardiac phenotype of PFHBI patients. Shown are electrocardiographic examples of PFHBI. (A) Sinus rhythm with a RBBB in an 8-year-old asymptomatic boy on a standard 12-lead ECG, with leads Std I, V1, and V6 shown. (B) Shown is 2:1 atrioventricular node block (atrial rate, 76 bpm; ventricular rate, 38 bpm) with a broad QRS complex on Holter monitoring in a 54-year-old man who had recently become symptomatic. ECGs were recorded at a 25 mm/s paper speed and 10 mm/mV signal amplitude.

Here, we further refined the genetic interval for the PFHBI disease locus and showed that a missense mutation in TRPM4 is the cause of blunted cardiac conduction in several branches of a large Afrikaner family. TRPM4 encodes a Ca²⁺-activated nonselective cation (CAN) channel in in vitro expression systems (11). Cardiac CAN channel activity has been suggested to contribute to the transient inward current (I_ti) initiated by Ca²⁺ waves. I_ti was described on Purkinje fibers, atrial and ventricular cardiomyocytes, and sinoatrial node cells (12). The identity of I_ti is controversial, but it appears to reflect 3 Ca²⁺-dependent components: Na⁺/Ca²⁺ exchange, Ca²⁺-activated chloride current, and current mediated by CAN channels such as TRPM4 (previously referred to as TRPM4b; refs. 12, 13). The PFHBI-associated mutation, which results in an amino acid sequence change in the TRPM4 N terminus, led to constitutive SUMOylation of TRPM4 and impaired TRPM4 endocytosis, resulting in a dominant gain of TRPM4 channel function. Our findings indicate a key role for the TRPM4 CAN channel in cardiac conduction.

PFHBI disease is linked to a mutation in TRPM4. We extended the clinical and genetic basis of our previous mapping and linkage study in the Afrikaner pedigree with PFHBI, traced to an ancestral founder who immigrated from Portugal to South Africa in 1696 (5). Of 71 mutation carriers identified, 48 had PMs implanted. For some family members with PMs, we had access to ECGs prior to the date of PM implantation in which they still had normal atrioventricular conduction. Of the combined individuals not in need of PMs and patients with normal atrioventricular conduction prior to implantation, 19 patients had RBBB, 8 had RBBB with left anterior or left posterior hemiblock, and 7 had no ECG abnormalities (Supplemental Table 1; supplemental material available online with this article; doi: 10.1172/JCI38292DS1). For the 19 patients with RBBB, heart rates, mean P-wave duration, and mean PR interval were in the normal range (Supplemental Table 1). Atrial premature activity and extra systoles were not seen. None of the characterized patients showed a Brugada syndrome type I pattern in ECG analysis, and syncope caused by Torsades de pointes was not observed in these families. It should be noted that with bundle branch blocks, QTc may be prolonged because of late depolarization, and consequent late repolarization, of one ventricle.

With additional microsatellite markers, we narrowed the PFHBI locus to an interval between markers D19S1059 and D19S604 corresponding to a region of approximately 0.5 Mb in chromosome 19q13.33 with approximately 25 genes (Supplemental Figure 1). Of these 25 genes, 5 are expressed in cardiac tissue: U1 small nuclear ribonucleoprotein (SNRP70); histidine-rich calcium-binding protein (HRC); transient receptor potential cation channel, subfamily M, member 4 (TRPM4); transcriptional enhancer 4 (TEAD2); and potassium voltage-gated channel, Shaker-related subfamily, member7 (KCNA7) (11, 14–18). Previously, KCNA7 was excluded as a possible candidate gene (16). Here, we focused on TRPM4 (11), which was prominently expressed in human Purkinje fibers compared with septum, atrium, and right and left ventricles (Figure 2A). We sequenced the 25 exons of TRPM4 (Supplemental Table 2). DNA samples of 23 affected and 35 unaffected members of the Afrikaner pedigree with PFHBI were available for analysis. We detected exclusively in DNA samples of affected family members a heterozygous G→A mutation at nucleotide 19 in exon 1 (c.19G→A; Figure 2B). The mutation generated a new recognition site for the restriction enzyme MboII, allowing for independent identification of the c.19G→A TRPM4 mutation by MboII DNA digestion (Supplemental Figure 2). In 2 unaffected control populations (230 ancestry-matched, unrelated Afrikaner and 389 unrelated individuals of mixed European descent), we did not detect the 19A allele. The c.19G→A mutation in TRPM4 predicts the substitution of TRPM4 glutamic acid at position 7 to lysine (p.E7K) within the TRPM4 N terminus (referred to herein as TRPM4^E7K). Glu7 is part of an N-terminal TRPM4 sequence motif evolutionary conserved among TRPM4 orthologs across phyla (Figure 2C). The motif is likely to play an important function in TRPM4 channel activity unrelated to conserved sequence domains previously reported to affect TRPM4 channel assembly or activity (Figure 2D and ref. 19).

Figure 2

TRPM4 missense mutation in exon 1 associated with PFHBI. (A) Relative expression of TRPM4 transcripts in different tissues of nondiseased human heart was assayed by quantitative RT-PCR. TRPM4 mRNA expression levels were normalized to the level in left ventricle. Numbers of individual probes are shown in parentheses. Each experiment was done in triplicate. (B) Electropherograms show TRPM4 WT sequence and the heterozygous sequence change c.19G→A in the DNA of PFHBI-affected individuals. (C) Partial amino acid sequence alignment of TRPM4 N terminus among different species. Gray shading shows the conserved sequence motif; red shading highlights the glutamic acid substituted by lysine in TRPM4 associated with PFHBI. Numbering refers to the human sequence. (D) Diagram of TRPM4 topology and functional domains, with 6 membrane-spanning domains (TM) flanked by N- and C-terminal cytoplasmic sequences. PFHBI, PFHBI domain; CaM, Calmodulin-binding domain; WB, Walker B ATP-binding motif; CCR, coiled-coiled region. Figure part adapted with permission from Pflügers Archiv (19).

The PFHBI-associated mutation increases TRPM4 current density. To understand the functional significance of the PFHBI-associated mutation, we expressed WT and mutant TRPM4 channels in HEK 293 cells and measured TRPM4 current properties in response to a variety of stimuli known to influence TRPM4 channel gating (11, 19). WT and mutant TRPM4 channels responded in an essentially identical manner to both change in membrane voltage and rise in intracellular Ca²⁺ (Figure 3, A and B). At +80 mV, half-maximal current activation was observed, with 0.44 ± 0.03 and 0.40 ± 0.04 μM Ca²⁺ for WT and mutant TRPM4 channels, respectively (n = 7 per group; Figure 3B), in agreement with data previously reported for the TRPM4 channel expressed in HEK 293 cells (11). WT and mutant TRPM4 channel exhibited essentially identical sensitivities to block by intracellular ATP in inside-out membrane patches. Bath application of 500 μM adenylyl imidodiphosphate tetralithium salt (AMP-PNP), a nonhydrolizable ATP analog, inhibited WT and mutant TRPM4 channels with similar potency (TRPM4, 72.5% ± 2.5% block; TRPM4^E7K, 71.3% ± 3.9% block, n = 3; Figure 3C, Supplemental Figure 3A, and ref. 20). Furthermore, application of phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P₂] to inside-out patches stimulated TRPM4 and TRPM4^E7K current amplitudes to a similar degree (TRPM4, 232% ± 12%, TRPM4^E7K, 238% ± 15%, n = 3; Supplemental Figure 3, B and C). These data indicated that PtdIns(4,5)P₂ sensitivity of TRPM4^E7K channels was unchanged compared with WT channels (21).

Figure 3

Expression of human TRPM4 and TRPM4^E7K in HEK 293 cells. Unless otherwise indicated, black traces denote TRPM4; red traces denote TRPM4^E7K. (A) Normalized current-voltage relationship for TRPM4 and TRPM4^E7K obtained from 250-ms voltage ramps measured in the whole-cell patch-clamp configuration from –120 to +100 mV. Holding potential was –60 mV. (B) Ca²⁺ dependence of TRPM4 current densities (I/I_max) obtained from voltage ramps measured at –80 and +80 mV (n = 7–16). [Ca²⁺]_i, intracellular Ca²⁺. (C) AMP-PNP block of WT TRPM4 and TRPM4^E7K current. Holding potential was 0 mV. Currents, elicited by a 250-ms pulse to +100 mV after a 500-ms pulse to –100 mV, were recorded before (0; black) and after application of 500 μM AMP-PNP (0.5; red) Scale bars: 200 ms, 0.5 nA. (D) Normalized current densities (I/I_norm) of TRPM4^E7K expressed alone (n = 13) or in a 1:1 ratio with TRPM4 (WT/E7K, n = 6) obtained from voltage ramps measured at +40 and +80 mV. n = 16 (WT TRPM4). *P < 0.05 versus WT. (E) Single-channel currents recorded from inside-out patches at –100 mV. Scale bars: 1 s, 5 pA. (F) Histogram plots of TRPM4 and TRPM4^E7K traces shown in E. (G) P_o of TRPM4 and TRPM4^E7K channel at +100 mV (n = 8–9).

However, comparison of TRPM4 channel densities showed that HEK 293 cells expressing TRPM4^E7K consistently exhibited approximately 2-fold TRPM4 current density (43.8 ± 6.3 pA/pF at +80 mV, n = 13) compared with that of cells expressing WT channel (18.8 ± 3.7 pA/pF at +80 mV, n = 10). We obtained identical results using CHO cells (Supplemental Figure 4). Furthermore, HEK 293 cells transfected with TRPM4^E7K cDNA or cotransfected with equal amounts of TRPM4 and TRPM4^E7K cDNA (43.9 ± 5.2 pA/pF at +80 mV, n = 6) showed essentially the same increase in current density (Figure 3D). The dominant mutational effect on TRPM4 current amplitude apparently reflects the dominant inheritance of the PFHBI phenotype.

Macroscopic current amplitude I is the product of 3 parameters: single-channel conductance (γ), open probability (P_o), and number of channels (N) expressed at the cell surface. First, we investigated in the inside-out patch-clamp configuration parameters γ and P_o, biophysical parameters intrinsic to the channel. They were essentially identical for WT (γ, 19.1 ± 0.8 pS; P_o, 0.25 ± 0.09, n = 8) and TRPM4^E7K channels (γ, 18.8 ± 0.4 pS; P_o, 0.26 ± 0.02, n = 9; Figure 3, E–G). The data indicate that the observed mutational effect on TRPM4 current amplitude was most likely the result of a change in N.

The PFHBI-associated mutation increases TRPM4 channel density. We addressed this hypothesis directly and evaluated the number of WT and mutant TRPM4 channels residing in the plasma membrane. In order to evaluate N, we introduced a Myc tag into an extracellular site of TRPM4 between the first and second transmembrane-spanning segments, according to the predicted membrane topology of TRPM4 (Supplemental Figure 5 and ref. 19). Functional tests showed that Myc-tagged WT and mutant TRPM4 channels mediated currents indistinguishable from the untagged channel (Supplemental Figure 6, A and B). Next, we labeled nonpermeabilized HEK 293 cells expressing Myc-tagged WT or TRPM4^E7K channels with FITC-coupled anti-Myc antibody and quantitated N using fluorescence-activated cell sorting (FACS; Figure 4A). HEK 293 cells expressing untagged WT channel were used for background control. In agreement with our electrophysiological results, the FACS results showed approximately 2.5-fold larger N for Myc-tagged TRPM4^E7K than for WT TRPM (Figure 4, A and B).

Figure 4

Analysis of TRPM4 and TRPM4^E7K protein density. (A) FACS fluorogram of nonpermeabilized cells expressing untagged TRPM4 control, Myc-tagged TRPM4, or Myc-tagged TRPM4^E7K labeled with FITC-conjugated Myc-specific antibody. FI, fluorescence intensity. (B) FACS analysis of fluorograms in A. n = 9 per group. (C–E) Cells expressing empty vector control (Vector), FLAG-tagged TRPM4, or FLAG-tagged TRPM4^E7K were fixed and permeabilized 48 hours after transfection. (C) Cells were stained with mouse FLAG-specific primary antibody and anti-mouse goat Alexa Fluor 546–coupled secondary antibody. Nuclei were stained with DAPI. Original magnification, ×20. (D) Immunofluorescence intensity of FLAG-tagged TRPM4^E7K normalized to that of FLAG-tagged TRPM4–expressing cells (n = 60 per group). (E) SDS-PAGE of C-terminally FLAG-tagged TRPM4 and TRPM4^E7K protein in cell lysates followed by Western blotting. Membranes were stained with mouse FLAG-specific antibody and simultaneously with rabbit actin-specific antibody for loading control. *P < 0.05, **P < 0.01.

A likely explanation for increased TRPM4^E7K channel density in the plasma membrane was a mutational influence on TRPM4 protein stability. To address this hypothesis, we investigated the steady-state concentration of TRPM4 WT and mutant channel protein in transiently transfected cells by Western blot and immunofluorescence microscopy. We used FLAG-tagged channels with current density similar to that of untagged controls (Supplemental Figure 6, C and D) to analyze TRPM4 protein concentration in transfected cells. Western blot analysis of respective cell lysates revealed a more intense signal for FLAG-tagged TRPM4^E7K than for WT (n = 3; Figure 4E). Fluorescence image quantitation (see Methods) revealed significantly larger immunofluorescence intensity for TRPM4^E7K than for WT (Figure 4, C and D). Thus, the effect of the PFHBI mutation on TRPM4 channel density correlated with an elevated steady-state level of TRPM4 protein.

The PFHBI-associated mutation affects TRPM4 channel endocytosis. The observed TRPM4^E7K concentration increase in the plasma membrane indicated an altered balance between the anterograde and retrograde TRPM4 trafficking pathways that determine steady-state protein expression at the cell surface. Because mutations mostly obstruct protein function (22–27), we considered a default in retrograde (i.e., endocytotic) TRPM4 trafficking as a likely explanation for the PFHBI mutational effect. Dynein-based endocytotic trafficking, a common pathway in ion channel endocytosis (28–30), is effectively inhibited in cells that overexpress dynamitin (31, 32). Overexpression of dynamitin is an established method to disrupt the dynein motor system and to interfere with ion channel endocytosis. Thus, we investigated the effect of dynamitin overexpression on TRPM4 channel density. Cotransfection of HEK 293 cells with dynamitin and TRPM4 channel cDNAs resulted in significantly increased TRPM4 channel density (39.3 ± 7.9 pA/pF, n = 9; Figure 5A). Conversely, TRPM4^E7K current density was dynamitin insensitive (42.7 ± 8.7 pA/pF, n = 7; Figure 5A). We conclude that the PFHBI mutation impaired dynein-based TRPM4 channel endocytosis.

Figure 5

Posttranslational regulation of TRPM4 and TRPM4^E7K channel density. (A) TRPM4 and TRPM4^E7K channel, expressed together with or without dynamitin (Dyn) for 18 hours in HEK 293 cells. Current densities were obtained at +80 mV as described in Figure 3A. n = 7 (E7K + Dyn); 9 (WT and WT + Dyn); 10 (E7K). (B) HEK 293 cells expressing Myc-tagged TRPM4 or TRPM4^E7K channel were incubated with or without 10 μM MG132. FACS analysis was performed as in Figure 4A. n = 3–9. *P < 0.05.

Endocytosis is the initial step in retrograde movement of membrane protein. Subsequently, internalized proteins can follow multiple routes with different outcomes. One well-recognized fate of internalized protein is targeting for proteasomal degradation (33). Therefore, we reasoned that WT TRPM4 is more sensitive to proteasomal degradation than is TRPM4^E7K, for which endocytosis is disrupted. To test this hypothesis, we investigated the effect of the proteasome inhibitor MG132 (34) on cell surface expression of WT and mutant Myc-tagged TRPM4 channels. Quantitative FACS analysis of Myc-specific antibody–labeled transfected HEK 293 cells showed that MG132 produced a significant, approximately 3-fold increase in channel density for WT (310% ± 30%, n = 3), but not TRPM4^E7K (Figure 5B). This finding concurs with our observation that the endocytotic TRPM4 trafficking was impaired by PFHBI mutation, possibly by its interference with a regulatory pathway.

The TRPM4^E7K channel is less sensitive to SUMOylation. The regulatory pathway presumably involves posttranslational TRPM4 modification, for example, by protein phosphorylation and/or dephosphorylation. In keeping with this idea, we applied 8-Brc-AMP to TRPM4-expressing HEK 293 cells to stimulate protein kinase A and then studied the effects of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine and the protein phosphatase inhibitor okadaic acid on TRPM4 current density. Both WT and TRPM4^E7K current density was insensitive to this treatment (Supplemental Figure 7). As an alternative posttranslational modification, one that may regulate ion channel density in the plasma membrane, we considered SUMOylation (33). First, we directly investigated SUMOylation of FLAG-tagged TRPM4. FLAG-tagged WT or mutant TRPM4 protein was pulled down with immobilized FLAG-specific antibodies from cellular lysates and immunostained with small ubiquitin-like modifier–specific (SUMO-specific) antibodies in Western blots. We found that TRMP4 protein was directly SUMOylated, whereas vector controls showed no detectable SUMOylation signal; moreover, TRPM4^E7K protein displayed markedly stronger immunostaining than did WT protein (n = 3 per group; Figure 6A).

Figure 6

Sensitivity of TRPM4 current density to SUMOylation. (A) C-terminally FLAG-tagged TRPM4 or TRPM4^E7K protein pulled down on beads coated with FLAG-specific antibodies were subjected to SDS-PAGE and Western blotting. Blots were stained with rabbit SUMO-1–specific primary antibody and anti-mouse rabbit horseradish peroxidase–coupled secondary antibody. Arrow indicates SUMOylated TRPM4. (B) Same experiment as in Figure 5A, but TRPM4 channel was coexpressed with Ubc9, SENP1, or the inactive mutant SENP1-C603S (SENP1*). n = 6–13. (C) Myc-tagged TRPM4^E7K was expressed in HEK 293 cells alone or together with Ubc9 or SENP1. At 24 hours after transfection, the surface density of Myc-tagged TRPM4^E7K channel was assessed by FACS analysis, as described in Methods. n = 3 per group. *P < 0.05.

Next, we investigated the effect of the SUMO ligase ubiquitin-conjugating enzyme 9 (Ubc9; ref. 35) and of the SUMO protease SUMO1/sentrin specific peptidase 1 (SENP1; ref. 36) on TRPM4 current density. Coexpression of WT TRPM4 with Ubc9 produced a significant increase of TRPM4 current density (43.3 ± 12.7 pA/pF, n = 9, P = 0.04). Conversely, coexpression of WT TRPM4 with SENP1 decreased TRPM4 current density (4.5 ± 1.0 pA/pF, n = 8) to background levels measured on mock-transfected cells (3.5 ± 0.4 pA/pF, n = 7, P = 0.39), whereas coexpression with the inactive SENP1 mutant SENP1-C603S (37) showed no effect on WT TRPM4 current density (21.6 ± 3.8 pA/pF, n = 9, P = 0.79; Figure 6B). In another control experiment, we investigated the effect of SENP1 protease on human ether-a-go-go–related gene (HERG) channel activity, which plays an important role in ventricular action potential repolarization (38). HERG current density was insensitive to SENP1 (absence, 53.7 ± 10.6 pA/pF, n = 6; presence, 54.8 ± 11.2 pA/pF, n = 7). In contrast to WT TRPM4, coexpression of TRPM4^E7K with Ubc9 (63.3 ± 7.1 pA/pF, n = 17, P = 0.46) or SENP1 (64.5 ± 17.2 pA/pF, n = 6, P = 0.65; Figure 6B) neither significantly increased nor significantly decreased current density. Quantitative FACS analyses in which we investigated surface expression of Myc-tagged TRPM4^E7K after coexpression with Ubc9 or SENP1 yielded similar results (Figure 6C). These results imply that deSUMOylation serves as a signal for TRPM4 internalization and that, accordingly, constitutive SUMOylation protects TRPM4^E7K against endocytosis and proteasomal degradation.

Here, we have identified a TRPM4 mutation in as the cause for an autosomal-dominant form of PFHBI in an Afrikaner pedigree, one of the largest reported families with chromosomal assignment (5). The mutation, TRPM4^E7K, caused a dominant TRPM4 gain of function. TRPM4^E7K correlated with elevated density of the TRPM4 channel in the plasma membrane because of attenuated channel deSUMOylation, a mechanism in the etiology of cardiac channelopathies (22, 39) that was, to our knowledge, previously unknown. Gain-of-function mutations in ion channels associated with cardiac arrhythmias are relatively rare (40–42). Ion channel mutations mostly lead to loss of channel function by disrupting subunit assembly, anterograde trafficking, and/or ion channel gating properties (22–27). The TRPM4^E7K channel, however, displayed normal gating properties — for example, γ, P_o, and regulation by intracellular Ca²⁺, PtdIns(4,5)P₂, or ATP — but increased expression at the cell surface as a result of a default in TRPM4 endocytosis. PFHBI (5) and ICCD (6) have been mapped on the same chromosomal locus and have a similar cardiac pathophysiology. It will be interesting to see whether ICCD is also associated with TRPM4 mutations increasing TRPM4 channel density at the cell surface.

It has become apparent that SUMO and ubiquitin pathways communicate and jointly affect the properties of common substrate proteins, often having an antagonistic relationship (33). Our data showed that the expression of WT TRPM4 at the cell surface was sensitive to deSUMOylation by SENP1. In contrast, expression of TRPM4^E7K at the cell surface was insensitive to deSUMOylation. We therefore conclude that constitutive SUMOylation of TRPM4^E7K is responsible for the elevated expression of the channel at the cell surface. It is possible that SUMOylation of TRPM4 interferes with TRPM4 ubiquitination, an important means to target protein for internalization and proteasomal degradation, and thereby protects TRPM4^E7K against internalization and proteasomal degradation. Intriguingly, disruption of ubiquitin-dependent internalization has been previously demonstrated for a mutation within the gene encoding the ENaC channel; this mutation is associated with Liddle syndrome, a dominant hereditary form of hypertension (30).

TRPM4 and TRPM5 channels are unique candidates for the CAN channel. They are equally permeable to Na⁺ and K⁺, but, in contrast to all other TRP channels, they are not permeable to Ca²⁺ (11, 43). TRPM4 channel properties resemble those of the native cardiac CAN channel NSC_Ca (13, 19, 44). The importance of CAN channel activity and its contribution to the Ca²⁺-dependent I_ti in the heart has been discussed for some time, but the physiological role in cardiac function is not well understood (13, 19, 44–47). Our data offer a direct approach toward the physiological role of the CAN channel TRPM4. Here we showed that TRPM4 was prominently expressed in Purkinje fibers and played an important role, which we believe to be previously unrecognized, in the human cardiac conduction system. It is likely that elevated expression of the TRPM4^E7K channel increases membrane leak conductance, disabling action potential propagation down the Purkinje fibers. The hypothesis is consistent with the occurrence of broadened QRS complex in the patient’s ECG, and could also explain the PFHBI phenotype of RBBB, bifascicular block, and heart conduction block. However, the progressive loss of ventricular conduction ability in PHFBI remains to be explored. Tentatively, chronic increase in leak conductance causes cell death, which may explain the replacement fibrosis observed postmortem in the His-Purkinje fiber system of a PFHBI patient (P. Brink, unpublished observation).

In other forms of inherited cardiac conduction disorders, mutations within cardiac Na⁺ or Ca²⁺ channel genes have been identified (48–51). They are associated with loss of ion channel function as well as with reduction in depolarizing inward currents and driving force for myocellular Ca²⁺ entry. It has been proposed that Ca²⁺-sensitive transient TRPM4-mediated inward current causes membrane depolarization and thereby limits the driving force for Ca²⁺ entry in excitable cells (19, 44, 47). Whether increased cardiac TRPM4 channel activity blunts cardiac conduction because of its effects on membrane depolarization and Ca²⁺ influx needs to be explored. Furthermore, increased TRPM4 channel activity may also influence activity of other ion channels, for example, recovery of voltage-gated ion channels from inactivation and activation of hyperpolarization-activated cation channels. The identification of TRPM4^E7K in patients with PFHBI paves the way for further characterization of the pathophysiology of conduction disease and may ultimately lead to less invasive therapy than the currently approach of timely PM implantation. The nature of the mutation also suggests that modulation of TRPM4 activity using Ca²⁺ or K⁺ channel inhibitors could present an avenue for treatment.

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The authors thank the participating families; A. Goosen for logistical support; L. Hofmeyer for updating ECG information; U. Ravens and E. Wettwer for providing heart tissue samples; G. Sachse for critical discussion; K. Steinbach for assistance with FACS analysis; and M. Raetz, K. Hergarten, M. Keuthen, and E. Deravanessian for excellent technical and logistical help. This study was supported by grants from the South African Medical Research Council and the Harry and Doris Crossley Foundation to P. Brink and V. Corfield. O. Pongs and E. Schulze-Bahr are supported by Deutsche Forschungsgemeinschaft grants Po 137/37-2 (FOR 604), Po 137/39-1 (FOR 885), and DFG 653/Ki13-1 and 13-2; by the Fonds der Chemischen Industrie, and by the Interdisziplinäres Zentrum für Klinische Forschung (Medical Faculty of the University of Münster).

Address correspondence to: Olaf Pongs, Falkenried 94, 20251 Hamburg, Germany. Phone: 49-40-7410-55082; Fax: 49-40-7410-55102; E-mail: morin@zmnh.uni-hamburg.de.

Conflict of interest: The authors have declared that no conflict of interest exists.

Reference information: J. Clin. Invest.119:2737–2744 (2009). doi:10.1172/JCI38292.

Alf Beckmann’s present address is: MVZ Dortmund, Dortmund, Germany.

See the related articles at Endocytic control of ion channel density as a target for cardiovascular disease and Extracellular K⁺ concentration controls cell surface density of I_Kr in rabbit hearts and of the HERG channel in human cell lines.

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