Impaired endocytosis of the ion channel TRPM4 is associated with human progressive familial heart block type I (original) (raw)
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 _Mbo_II, allowing for independent identification of the c.19G→A TRPM4 mutation by _Mbo_II 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 TRPM4E7K). 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).
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; TRPM4E7K, 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 TRPM4E7K current amplitudes to a similar degree (TRPM4, 232% ± 12%, TRPM4E7K, 238% ± 15%, n = 3; Supplemental Figure 3, B and C). These data indicated that PtdIns(4,5)P2 sensitivity of TRPM4E7K channels was unchanged compared with WT channels (21).
Expression of human TRPM4 and TRPM4E7K in HEK 293 cells. Unless otherwise indicated, black traces denote TRPM4; red traces denote TRPM4E7K. (A) Normalized current-voltage relationship for TRPM4 and TRPM4E7K 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/Imax) 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 TRPM4E7K 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/Inorm) of TRPM4E7K 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 TRPM4E7K traces shown in E. (G) Po of TRPM4 and TRPM4E7K channel at +100 mV (n = 8–9).
However, comparison of TRPM4 channel densities showed that HEK 293 cells expressing TRPM4E7K 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 TRPM4E7K cDNA or cotransfected with equal amounts of TRPM4 and TRPM4E7K 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 (Po), and number of channels (N) expressed at the cell surface. First, we investigated in the inside-out patch-clamp configuration parameters γ and Po, biophysical parameters intrinsic to the channel. They were essentially identical for WT (γ, 19.1 ± 0.8 pS; Po, 0.25 ± 0.09, n = 8) and TRPM4E7K channels (γ, 18.8 ± 0.4 pS; Po, 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 TRPM4E7K 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 TRPM4E7K than for WT TRPM (Figure 4, A and B).
Analysis of TRPM4 and TRPM4E7K protein density. (A) FACS fluorogram of nonpermeabilized cells expressing untagged TRPM4 control, Myc-tagged TRPM4, or Myc-tagged TRPM4E7K 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 TRPM4E7K 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 TRPM4E7K normalized to that of FLAG-tagged TRPM4–expressing cells (n = 60 per group). (E) SDS-PAGE of C-terminally FLAG-tagged TRPM4 and TRPM4E7K 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 TRPM4E7K 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 TRPM4E7K than for WT (n = 3; Figure 4E). Fluorescence image quantitation (see Methods) revealed significantly larger immunofluorescence intensity for TRPM4E7K 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 TRPM4E7K 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, TRPM4E7K 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.
Posttranslational regulation of TRPM4 and TRPM4E7K channel density. (A) TRPM4 and TRPM4E7K 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 TRPM4E7K 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 TRPM4E7K, 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 TRPM4E7K (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 TRPM4E7K 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 TRPM4E7K 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, TRPM4E7K protein displayed markedly stronger immunostaining than did WT protein (n = 3 per group; Figure 6A).
Sensitivity of TRPM4 current density to SUMOylation. (A) C-terminally FLAG-tagged TRPM4 or TRPM4E7K 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 TRPM4E7K was expressed in HEK 293 cells alone or together with Ubc9 or SENP1. At 24 hours after transfection, the surface density of Myc-tagged TRPM4E7K 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 TRPM4E7K 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 TRPM4E7K 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 TRPM4E7K against endocytosis and proteasomal degradation.