TNNT2 (cardiac troponin T—TNNT2) are linked to increased risk of ventricular arrhythmogenesis and sudden death despite causing little to no cardiac hypertrophy. Therefore, studying the effect of TNNT2 variants on cardiac propensity for arrhythmogenesis can pave the way for characterizing HCM in susceptible patients before sudden cardiac arrest occurs. In this study, a TNNT2 variant, I79N, was generated in human cardiac recombinant/reconstituted thin filaments (hcRTF) to investigate the effect of the mutation on myofilament Ca2+ sensitivity and Ca2+ dissociation rate using steady-state and stopped-flow fluorescence techniques. The results revealed that the I79N variant significantly increases myofilament Ca2+ sensitivity and decreases the Ca2+ off-rate constant (koff). To investigate further, a heterozygous I79N+/−TNNT2 variant was introduced into human-induced pluripotent stem cells using CRISPR/Cas9 and subsequently differentiated into ventricular cardiomyocytes (hiPSC-CMs). To study the arrhythmogenic properties, monolayers of I79N+/− hiPSC-CMs were studied in comparison to their isogenic controls. Arrhythmogenesis was investigated by measuring voltage (Vm) and cytosolic Ca2+ transients over a range of stimulation frequencies. An increasing stimulation frequency was applied to the cells, from 55 to 75 bpm. The results of this protocol showed that the TnT-I79N cells had reduced intracellular Ca2+ transients due to the enhanced cytosolic Ca2+ buffering. These changes in Ca2+ handling resulted in beat-to-beat instability and triangulation of the cardiac action potential, which are predictors of arrhythmia risk. While wild-type (WT) hiPSC-CMs were accurately entrained to frequencies of at least 150 bpm, the I79N hiPSC-CMs demonstrated clear patterns of alternans for both Vm and Ca2+ transients at frequencies >75 bpm. Lastly, a transcriptomic analysis was conducted on WT vs. I79N+/−TNNT2 hiPSC-CMs using a custom NanoString codeset. The results showed a significant upregulation of NPPA , NPPB (brain natriuretic peptide), Notch signaling pathway components, and other extracellular matrix (ECM) remodeling components in I79N+/− vs. the isogenic control. This significant shift demonstrates that this missense in the TNNT2 transcript likely causes a biophysical trigger, which initiates this significant alteration in the transcriptome. This TnT-I79N hiPSC-CM model not only reproduces key cellular features of HCM-linked mutations but also suggests that this variant causes uncharted pro-arrhythmic changes to the human action potential and gene expression.Hypertrophic cardiomyopathy (HCM) is the most common heritable cardiovascular disease and often results in cardiac remodeling and an increased incidence of sudden cardiac arrest (SCA) and death, especially in youth and young adults. Among thousands of different variants found in HCM patients, variants of  MYH7) gene mutation in 1995, more than 1,000 different variants have been identified in over 20 HCM candidate genes (Hypertrophic cardiomyopathy (HCM) is a genetic disorder that presents clinically as ventricular and/or septal hypertrophy, myocyte disarray, and increased myocardial fibrosis . It is ate genes , most ofTNNT2) are the third most common cause of HCM after β-myosin heavy chain and myosin binding protein-C (MYBPC3)  variants typically show significantly less ventricular hypertrophy compared to other HCM-associated variants. This low degree of clinical penetrance of the TNNT2 variants makes them difficult to detect by echocardiography (TNNT2 variants (Variants found in the cardiac troponin T gene ((MYBPC3) . The HCMogenesis . Unlike iography , yet thevariants .TNNT2 variants were studied prior to the development of any overt form of hypertrophy   .2+-binding properties and the propensity for arrhythmias induced by a TNNT2 mutation, I79N, in human cardiac reconstituted thin filament (hcRTF) system and hiPSC-CMs.In this study, we investigated the altered Ca2+ sensitivity and dissociation using steady-state and stopped-flow fluorescence techniques, respectively.Both human recombinant cardiac troponin (cTn) complexes and hcRTF were used to determine the changes in Ca+/−TNNT2 hiPSC-CMs.In addition, we used hiPSC-CMs, which have been used to model a variety of inherited arrhythmias and cardiomyopathies . These h2+ sensitivity of the contractile element. Among the many different HCM-causing mutations, TNNT2 variants can be considered as one of the most critical, since they have a low degree of clinical penetrance. Studies in mice suggest that the HCM-associated TnT-I79N variant increases myofilament Ca2+ sensitivity and is highly arrhythmogenic, but whether findings from mice translate to human cardiomyocyte electrophysiology was not as well established. This is where the necessity of using human models to further investigate the I79N variant emerges.It is generally accepted that HCM-causing genetic variants increase the Ca2+-binding properties and the propensity for arrhythmias induced by a TNNT2 variant, I79N. Based on the modeling data, the arrhythmogenicity is related, at least in part, to the reduced off-rate constant (koff) of Ca2+ from the myofilaments. Action potential triangulation, which is purported to be arrhythmogenic, was clearly visible at low stimulation frequencies in I79N+/− hiPSC-CMs but was non-visible at higher frequencies, which elicited alternans. In addition, the mRNA expression profile strongly indicates that this TNNT2 variant induces a major remodeling response even with the cells not being fully mature, a finding which deserves further exploration.In this study, we used the hcRTF system and human-induced pluripotent stem cell-derived cardiomyocytes to investigate the altered CaTNNC1, TNNI3, and TNNT2). Reconstituted thin filaments were generated by mixing each cTn complex with human alpha-tropomyosin (TPM1) as well as native actin extracted from rabbit skeletal muscle, which has a 99.2% sequence identity with human cardiac actin (The adult cTn complexes were generated from recombinant human adult troponin subunits (expressed from ac actin . See Sup2 were added to 2 ml of each labeled hcRTF in a titration buffer containing the following (in millimolars): 200 MOPS, 150 KCl, 3 MgCl2, and 1 DTT at pH 7.0. At least five to eight datasets were collected for each construct, and the data were fit with a sigmoidal fit (based on the Hill equation) in Origin 8.5 . The Ca2+ sensitivity for each hcRTF was represented by the dissociation constant Kd (±SEM) defined as the Ca2+ concentration at the half-maximal fluorescence change. Two means were considered to be significantly different when p ≤ 0.05.All steady-state fluorescence measurements were performed using a Cary Eclipse fluorescence spectrophotometer (Agilent) at 15 ± 0.1°C as described . In brie2+ dissociation rates [koff(Ca2+)] from each hcRTF were characterized using a Chirascan stopped-flow apparatus  with a dead time of 1.1 ms at 15.0 ± 0.1°C. IAANS fluorescence was excited at 300 nm and monitored through a 510-nm broad band-pass interference filter . Each trace was obtained as the solution containing hcRTF saturated with 200 μM CaCl2 was rapidly mixed with 10 mM EGTA solution in the stopped-flow buffer containing the following(in millimolars): 10 MOPS, 150 KCl, 3 MgCl2, and 1 DTT at pH 7.0. Each koff(Ca2+) value represents an average of at least five traces fit with a single exponential equation and repeated more than 15 times. Two means were considered to be significantly different when p ≤ 0.05.Cahttps://www.wicell.org/). Differentiation of hiPSCs to cardiomyocytes (hiPSC-CMs) employed a protocol published previously  1640 basal medium  plus B27 without insulin supplement (Thermo Fisher) containing 12 µM CHIR99021 (R&D Systems) for 24 h, followed by 5 mM IWP4 (Tocris) for 2 days without medium change. The medium was changed to RPMI 1640 plus B27 complete supplement, and then subsequently, the medium was changed every 2–3 days. Robust spontaneous beating of the monolayer was observed by day 12.Human iPSC lines (iPS IMR90-1) were obtained from the Wicell Research Institute (eviously . In brieTNNT2 using online CRISPR tools including CRISPRko Tool from the Broad Institute (Single-guide RNAs (sgRNA) were designed to target the region close to I79 nstitute , the CRInstitute , and Bennstitute . The sgRnstitute . The pCCnstitute  were desnstitute  to inclunstitute .5 hiPSCs were transfected with 500 ng sgRNA and 10 pmol of ssODN. Since the vector contained GFP as a reporter, the GFP+ hiPSCs were sorted as single cells using fluorescence-activated cell sorting (FACS) 48–72 h post-transfection and were plated on Matrigel-coated plates in mTeSR1 (TNNT2 site using the primers described in Human iPSCs were co-transfected with a PX458 plasmid and ssODN using Lipofectamine 3000 (Thermo Fisher). Briefly, 2 × 10n mTeSR1 . More th2, 1 MgCl2, 10 HEPES, and 5.5 D-glucose for optical mapping as described (2+-sensitive dye Rhod-2AM (Molecular Probes) and 20 μM blebbistatin [a myosin ATPase inhibitor (Sigma-Aldrich)] were added to the culture dish. Blebbistatin inhibits contraction and reduces motion artifact with no detectable effect on electrophysiological properties (TNNT2 heterozygous mutation I79N+/− and wild type (WT) were stimulated for 20 s each at four stimulation frequencies . Data were analyzed using a custom-built software in IDL  : 140 NaCl, 3.6 KCl, 1.2 CaClescribed . In brieoperties . For imaean, VA) .2+ release units (CRUs). The model has been used to investigate spatiotemporal Ca2+ cycling and action potential dynamics in ventricular myocytes, including the genesis of Ca2+ waves and Ca2+ alternans  of Ca2+ from troponin C was reduced to 0.1 ms−1.Computer simulations were carried out to investigate the mechanisms of alternans promoted by the I79Nvariant. We used a ventricular cell model developed previously , consistlternans . Detailslternans , and theus model :Jtrpn=kQuantitative real-time PCR: Total RNA was extracted from hiPCS-CM using TRIzol followed by RNeasy kit , according to the manufacturer’s instructions. The concentration and purity of the RNA was determined using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific). The cDNA was synthesized using Qiagen Quantitect Reverse Transcription kit, according to the manufacturer’s instructions. Real-time qRT-PCR analysis was performed on a Bio-Rad CFX96 Touch Real-Time PCR System . Primers were designed using Primer3 and OligoAnalyzer 3.1. Analysis was performed according to the ΔΔCt method using the geometric mean of housekeeping genes β-actin and GAPDH to normalize the qRT-PCR data.Multiplexed mRNA profiling was conducted using a custom codeset containing 251 gene probes synthesized by NanoString Technologies Inc. and the NanoString nCounter® SPRINT Profiler28  in all analysis except for the differential expression of genes (DEG) analysis of the NanoString data. For DEG, the fold-change of each gene was compared between the WT and I79N+/−TNNT2 hiPSC-CMs using Welch’s t-test and was corrected for multiple comparisons using the Benjamini–Yekutieli method to calculate the false discovery rate (FDR). A p value <0.05 was considered statistically significant with the following notation: *p < 0.05, **p < 0.01, and ***p < 0.001.Data were presented as mean ± SEM unless noted otherwise. Statistical analysis and data visualization of hcRTF CaTNNT2 from residues 1 to 109, which are homologous sequences from mammals to teleosts. The conservation of the isoleucine at residue 79 represents the fact that is preserved for more than 400 million years of evolution.2+-dependent IAANS fluorescence increase was observed for all hcRTF constructs. The I79N variant shows a statistically significant increase of Ca2+ sensitivity by∼2.3-fold (p ≤ 0.05)  .2+ dissociation rates from the hcRTF constructs. Upon the addition of EGTA, the Ca2+ rapidly dissociated from cTnC, inducing a cTnC conformational change in which the fluorescence change was recorded. As Ca2+ was stripped from cTnC by EGTA, a decrease in IAANS fluorescence was observed for all hcRTF constructs. The hcRTF harboring the I79N TnT variant show a significant decrease of koff compared to WT, increasing the Ca2+ sensitivity by ∼1.4-fold (p ≤ 0.05) (2+ sensitivity conferred by the hcRTF harboring this TNNT2 variant in the steady-state experiments.Stopped-flow fluorescence spectroscopy was used to determine the Ca ≤ 0.05) . This exTNNI3/TNNI1 ratio were not significantly different between I79N+/− and WT lines  and cytosolic Ca2+ transients were determined from the fluorescence emitted by RH-237 and Rhod-2, respectively. Vm transients from hiPSC-CM monolayers stimulated at 55, 65, and 75 bpm. 30), and 80 as a function of stimulation frequency. 2+ transient duration at 30% recovery (CaTD30), and 80 as a function of frequency. The Vm and Ca2+ transients from both WT and I79N+/− hiPSC-CMs entrained normally to 55, 65, and 75 bpm stimulation frequencies. These frequencies were chosen because 55 bpm was higher than the intrinsic beating rate of all hiPSC-CMs and 75 bpm was below frequencies that induced any observable form of arrhythmia. The action potential morphology showed significant triangulation [(APD80 − APD30) / APD80] in I79N+/− compared to WT hiPSC-CMs at 55 but not at 65 and 75 bpm (data not shown).We next investigated the effect of various stimulation frequencies in WT and I79N30 and APD80 as the frequency is increased from 55 to 75 bpm, the same does not hold true for I79N+/− hiPSC-CMs. In contrast, the Ca2+ transient duration decreases significantly in both groups with increased stimulation frequency.Vm (red) and Ca2+ transients (black) at stimulation frequencies of 55, 65, and 100 bpm are shown in +/− hiPSC-CMs entrained well to frequencies up to 75 bpm. However, at stimulation frequencies greater than 75 bpm, the arrhythmogenicity of the I79N+/− lines is clearly demonstrated with an alternans pattern seen for both voltage and Ca2+ transients. In stark contrast, WT hiPSC-CMs could be entrained normally to stimulation frequencies >150 bpm (data not shown).Representative optical mapping traces of koff = 0.5 ms−1) and slowed (koff = 0.1 ms−1) Ca2+ off rate constants are shown in 2+ concentration , no alternans was present for PCLs from 300 to 700 ms (black dots in koff(Ca2+), alternans occurred when the PCL was shorter than 500 ms (>120 bpm)  showing a strong clustering to TNNT2 genotype. In PCA, the hiPSC-CM samples were clustered according to the TNNT2 genotype with samples possessing the I79N+/− or WT TNNT2 being strongly associated to samples of the same TNNT2 genotype. The principal component 1 (PC1) and PC2 accounted for 73% and 16% of the total variance, respectively. The high variance observed in PC1 demonstrated that the TNNT2 mutation variability  is the largest driver of the difference at the gene expression level.To characterize further the effects of the I79N codeset , 236 geniPSC-CMs . The heavia the volcano plot, revealed 68 genes differentially expressed  between the I79N+/− variant and WT hiPSC-CMs , which encodes for atrial natriuretic peptide, and NPPB (5.9-fold), which encodes for brain natriuretic peptide. In addition, Notch signaling components  were significantly (p < 0.001) upregulated in I79N+/− compared to WT. Transcripts encoding for the components of collagen  were also dramatically upregulated in I79N+/− hiPSC-CMs. Significantly downregulated transcripts included SOX2 and cardiac developmental marker BMP4. The ratio of TNNI3/TNNI1, which is used as an index of hiPSC-CM maturation, was 6.1 and 5.6 in I79N+/− and WT, respectively, and was not significantly different between the groups.DEG analysis, illustrated iPSC-CMs . In the +/− and WT samples. The top five functional pathways that were found to be significantly dysregulated  across six gene set libraries are highlighted in +/− include Notch signaling, both canonical and non-canonical, which includes the maturation precursor as well as the Notch receptor-ligand signaling  associated to 45 upregulated DEGs in I79N+/−  enriched in terms associated with the regulation of cardiac muscle cell membrane repolarization , and the changes in transcriptome. Our results showed that I79N caused Ca2+ mishandling patterns that are consistent with proarrhythmic signals and dysregulated transcriptomic signaling pathways, which further elucidated the cellular mechanisms behind complex cardiomyopathy phenotypes of I79N that lead to sudden cardiac death.Despite over 20 years of studies conducted on HCM-causing TNNT2, most regions remain unsolved except for regions including residues 199–272 that is seen in the high-resolution crystal structure (PDB: 4Y99)  for I79N due to the lack of the structural information in this region of the molecule.Due to the highly flexible nature of B: 4Y99) . However+/−TNNT2 variant are associated with a high rate of sudden cardiac death at a young age , potentially resulting in triggered activity or delayed after depolarization. In addition, a heterozygous I79N variant was found in a family with nine affected members and caused different phenotype characteristics of RCM, HCM, DCM, and mixed cardiomyopathy within the same family  to generate heterozygous I79N+/−TNNT2 hiPSCs. This technique enabled us to compare I79N+/−TNNT2 hiPSCs with its isogenic control hiPSCs instead of comparing with control hiPSCs that have different genetic and epigenetic backgrounds. It has been shown that I79N-related HCM cases are characterized by mild hypertrophy , the rapid heart rate resulted in atrial fibrillation or sinus tachycardia in over 90% of the arrhythmic episodes ] from troponin C during diastole free during systole and leads to a shorter plateau phase in APD. Moreover, the downregulation of the inward rectifier current (Ik1) in I79N transgenic mice contributes to the shortening of the APD free during systole was reduced due to increased Ca2+ sensitivity, the activity of the NCX decreased, resulting in a smaller NCX current and a shorter plateau phase  during fast pacing may lead to arrhythmogenic events.It has been reported that the Canic mice . Previou the APD . Our finng assay . One posactivity . The priau phase . The rolmyocytes . Howevermyocytes . The AP hmogenic  and can hmogenic . However2+ alternans. It was called it the “3R” theory since Ca2+ alternans is promoted by the interactions of random firing of the Ca2+ sparks, recruitment of neighboring Ca2+ CRUs for firing (spark-induced sparks), and refractoriness of the CRUs. The recruitment is determined by the coupling strength between CRUs, and Ca2+ buffering is one of the parameters affecting recruitment  increases the cytosolic Ca2+ transient but reduces the SR Ca2+ load, indicating that slowing the Ca2+ dissociation rate caused more CRUs to fire due to the enhanced recruitment.In previous studies , we deveruitment . In thisruitment . This th+/−TNNT2 variant on the transcriptome profile demonstrating increased signaling of a hypertrophic state, profibrotic extracellular matrix (ECM) gene network, and the dysregulation of cardiac developmental pathways. The significant consequence of the I79N+/− variant on the transcriptome profile requires further investigation. Every precaution was taken to ensure, to the best of our ability, that the WT and I79N+/−TNNT2 variant hiPSC-CMs were as similar as possible except for the point mutation. Both lines were exposed to the CRISPR/Cas9 genome-editing protocol with the I79N+/− line undergoing a double-strand break and then homology-directed repair while the WT remain unchanged. The 10 most likely off-target mutation sites were all sequenced to determine that this was not a contributing factor. Both lines were then subjected to identical differentiation protocols in parallel, and RNA was isolated at the same time. Thus, the most likely explanations for the striking observation are the functional consequences of the variant of biophysical aspects of the contractile process and the alterations in hiPSC-CM Ca2+ handling due to the decrease in the Ca2+ off-rate constant from the myofibrils.We assessed the impact of the I79NThe significant increase in the expression of the transcripts for atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) is indicative of a remodeling taking place that likely reflects a hypertrophic response . MechaniNotch1, Notch3, Notch4, Jag1, and Hey1) were all significantly upregulated in I79N+/− hiPSC-CMs. The Notch signaling pathway plays an important role during development and maintaining homeostasis by regulating cellular apoptosis, proliferation, and differentiation as well as playing an important role in intercellular communication  (which was significantly upregulated in I79N+/− hiPSC-CMs) and one chain of collagen type I alpha 2 (COL1A2). The COL3A1 gene, which encodes for the collagen alpha-1 (III) chain, known as the alpha 1 chain of type III collagen, and was increased significantly in this study. Since the ECM network transmits mechanical signals to cardiomyocytes, disruption or excess production and accumulation of the ECM proteins observed in cardiac fibrosis can significantly affect contraction and relaxation as well as the architecture and function of the heart (TNNT2 variant I79N+/− induces a broad spectrum of adaptations.The modification of transcripts associated with collagen was also striking and indicative of a remodeling process. Collagen is the main structural protein of the cardiac ECM, which consists of approximately 85% type I collagen, which is responsible for building thick fibers and is important for strength, and 11% type III collagen, which assembles as thin fibers and plays an important role in ECM elasticity. The ECM also includes collagen types IV, V, and VI; fibronectin; laminin; elastin and fibrillin; proteoglycans; and glycoproteins . Collagehe heart . Althoug+/−TNNT2 hiPSC-CMs using CRISPR/Cas9 and optically mapped their physiological features including voltage and Ca2+ transients. Our findings indicate that the alteration of AP and Ca2+ handling in I79N+/−TNNT2 hiPSC-CMs is proarrhythmic at higher stimulation frequencies. Remarkably, the transcriptome was dramatically impacted by the insertion of a point mutation in one allele of troponin T and likely has profound consequences in a disease phenotype. The hiPSC-CMs along with the genome-editing tools demonstrate the utility of this technology for modeling sarcomeric mutations linked with HCM, identifying the arrhythmogenic factors and, potentially, developing therapeutic agents for the treatment of HCM.In conclusion, we generated I79N


TRPM7 channels are abundantly expressed in ameloblasts and, in the absence of TRPM7, dental enamel is hypomineralized. The potential role of TRPM7 channels in Ca2+ transport during amelogenesis was investigated in the HAT-7 rat ameloblast cell line. The cells showed strong TRPM7 mRNA and protein expression. Characteristic TRPM7 transmembrane currents were observed, which increased in the absence of intracellular Mg2+ ([Mg2+]i), were reduced by elevated [Mg2+]i, and were inhibited by the TRPM7 inhibitors NS8593 and FTY720. Mibefradil evoked similar currents, which were suppressed by elevated [Mg2+]i, reducing extracellular pH stimulated transmembrane currents, which were inhibited by FTY720. Naltriben and mibefradil both evoked Ca2+ influx, which was further enhanced by the acidic intracellular conditions. The SOCE inhibitor BTP2 blocked Ca2+ entry induced by naltriben but not by mibefradil. Thus, in HAT-7 cells, TRPM7 may serves both as a potential modulator of Orai-dependent Ca2+ uptake and as an independent Ca2+ entry pathway sensitive to pH. Therefore, TRPM7 may contribute directly to transepithelial Ca2+ transport in amelogenesis.TRPM7 plays an important role in cellular Ca The depletion of cellular Mg2+ is known to activate TRPM7-mediated currents [+ may have a net stimulatory effect [2+] and pH during enamel formation might therefore be expected to regulate TRPM7-dependent Ca2+ and Mg2+ uptake into the ameloblasts.Studies performed in native and heterologous expression systems indicate that TRPM7-mediated currents are regulated both by the intracellular Mgcurrents , and botcurrents ,20. In t2+ and pH changes. To explore this possibility, we have used the HAT-7 cell line, derived from rat incisor cervical loop  and pH, and the corresponding changes in intracellular Ca2+ concentration ([Ca2+]i) and pH (pHi).In the present study, we have measured mRNA and protein expression, ion currents evoked by TRPM7-specific activators and inhibitors during manipulation of extra- and intracellular i   by the TRPM7 inhibitor NS8593 (20 µM) (Changes in the intracellular Ca < 0.05) A,B. Thissolution A,B confi (20 µM) A,B.2+]i by 52 ± 5%   in the nominally Ca2+-free bath solution  (Stimulation with 50 µM mibefradil increased i by 12 ± 2% . However, applied under these conditions, both mibefradil (50 μM) and naltriben (100 μM) significantly enhanced the TRPM7-mediated Ca2+ influx compared to what was observed at normal pHi. The rise in [Ca2+]i evoked by mibefradil increased from 43 ± 6% at normal pHi to 78 ± 3.9% during the intracellular acidification  , indicating a marked stimulatory effect of intracellular protons on the activation of TRPM7. In this context it is interesting to note that a reduction in extracellular pH from 7.3 to 6.3, as applied in the whole-cell current recordings shown in n = 6, p < 0.05), as measured by BCECF fluorescence (The application of NH-7 cells C. Followy pH 6.4 C. Duringnditions D. This i < 0.05) D. Likewirescence E.2+ uptake pathway sensitive to changes in both extra- and intracellular pH.Taken together these results indicate the presence of functional TRPM7 channels in HAT-7 ameloblast cells. They also suggest that TRPM7 channels, in addition to their role in modulating SOCE, may serve as a CaIn this study we have shown that TRPM7 channels are expressed abundantly in HAT-7 cells, both at mRNA and protein levels. This finding is not surprising in light of the previous work showing that TRPM7 expression is very high in ameloblasts and odontoblasts as compared with other organs , and tha2+ depletion in HAT-7 cells elicited outward currents that were inhibited by the known TRPM7 antagonists NS8593 [2+]i. It is important to note that in our experiments the presence of divalent cations in the extracellular solution prevented the activation of the inward currents that have been observed by others i may cause small increases in Ca2+-activated K+ channel activity. Since our bath solution contained only 2.8 mM K+, we assume that the negative Erev values are mainly due to some Cs+ permeability of the K+ channels [+ currents.The measurement of the reversal potentials is difficult under these conditions because the current is nonlinear and has a shallow slope around Echannels  rather t2+ secretion into the enamel space by ameloblasts requires Ca2+ uptake at the basolateral membrane to balance Ca2+ efflux across the apical membrane. As seen in other cell types [2+ by HAT-7 cells was significantly stimulated by the TRPM7 activators. Naltriben evoked a marked increase in [Ca2+]i that was completely abolished in a nominally Ca2+-free environment, indicating that the TRPM7 channels can contribute to Ca2+ influx into HAT-7 cells. However, the SOCE inhibitor BTP2 strongly blocked the naltriben-induced Ca2+ entry, suggesting a pivotal role for Orai channels in these processes. This observation is in agreement with a previous report by Souza Bomfim et al. i. Importantly, the mibefradil-induced Ca2+ entry was not inhibited by BTP2, which implies that TRPM7 channels may provide a Ca2+ uptake pathway independent of SOCE. Our data suggest that mibefradil not only stimulated Ca2+ entry from the extracellular space, but also induced Ca2+ release from intracellular stores. Furthermore, we observed that mibefradil slightly reduced [Ca2+]i in cells pretreated with thapsigargin. This is consistent with recent studies showing that mibefradil reduces the Ca2+ leak from the ER following SERCA inhibition in endothelial and HK-2 epithelial cells i responses. Cells were illuminated alternately with excitation wavelengths of 340 and 380 nm using a metal-halide lamp and an internal filter wheel . Imaging was performed with a cooled CCD camera , coupled to the microscope and controlled by NIS AR software . Changes in [Ca2+]i were calculated as the ratio of the emitted fluorescence at the two respective excitation wavelengths (F340/F380) and normalized to the baseline ratio. Quantitative data were obtained by defining an ROI encompassing at least 50 cells.Cells cultured on coverslips were incubated with 4 μM fura-2 AM  in bath solution for 45 min at room temperature and washed prior to calcium imaging. The coverslips were mounted on an upright fluorescence microscope  in a custom-made open perfusion chamber and perfused with bath solution containing (in mM) 137 NaCl, 5 KCl, 2 CaCl+/nigericin method [Intracellular pH changes were monitored in real time by microfluorometry using the pH-sensitive fluorescent indicator BCECF as described previously . Brieflyn method .t-tests, planned pairwise comparisons using paired or unpaired two-sample t-tests as well as one-way ANOVA followed by Dunnett’s multiple comparison test; p < 0.05 was considered as significant.Data are presented as the mean ± SEM. Statistical analyses were performed with one-sample


SLC24A6/SLC8B1, coding the Na+/Ca2+/Li+ exchanger (NCLX), was identified as the gene responsible for mitochondrial Na+-Ca2+ exchange, a major Ca2+ efflux system in cardiac mitochondria. This molecular identification enabled us to determine structure–function relationships, as well as physiological/pathophysiological contributions, and our understandings have dramatically increased. In this review, we provide an overview of the recent achievements in relation to NCLX, focusing especially on its heart-specific characteristics, biophysical properties, and spatial distribution in cardiomyocytes, as well as in cardiac mitochondria. In addition, we discuss the roles of NCLX in cardiac functions under physiological and pathophysiological conditions—the generation of rhythmicity, the energy metabolism, the production of reactive oxygen species, and the opening of mitochondrial permeability transition pores.It has been over 10 years since  For the physiological functions of cardiomyocytes, it is vital for mitochondrial matrix Ca2+ to be maintained within an appropriate range because several metabolic enzymes are activated by Ca2+ to supply ATP, meeting cellular ATP demand, but excess Ca2+ causes mitochondrial dysfunction via opening of mitochondrial permeability transition pores (mPTP) . In 2+ handling system varies from tissue to tissue, which may contribute to tissue-specific tuning of mitochondrial as well as cellular functions. It is well accepted that CUmit activity is lower in the heart than in other tissues [mit activity may be due to differences in stoichiometry among a pore-forming protein (MCU), a dominant negative subunit (MCUb), and/or an EF-hand containing Ca2+-sensitive regulator (MICU1)—an MCUb:MCU ratio that is higher and an MICU1:MCU ratio that is lower in the heart [mit via a certain cytosolic Ca2+ threshold was not observed in heart, though it has been well described functionally and structurally in other tissues or cell types . These 2+ influx, NCXmit and H+-Ca2+ exchange (HCXmit) extrude Ca2+ from mitochondria, with the former accounting for the major component in excitable tissues such as the heart and brain, and the latter being dominant in non-excitable tissues such as the liver and kidney . Rystede review  quantita tissues ,18. Inte+-dependent Ca2+ efflux to total Ca2+ efflux is 60–100%, depending on species and experimental conditions [mit, though its contribution in the heart has been controversial. Leucine-zipper-EF hand-containing transmembrane (Letm1), which was initially shown to mediate H+-dependent Ca2+ influx into mitochondria [mit. Natarajan et al. [+-induced Ca2+ efflux from rat cardiac mitochondria, which were dependent on the free matrix Ca2+ concentration. Furthermore, they confirmed Letm1-mediated Ca2+ efflux from mitochondria by demonstrating a diminished Ca2+ efflux rate in permeabilized H9c2 cells due to Letm1 knockdown. Interestingly, they found that the expression level of the Letm1 protein in mitochondria was higher in the heart than in the liver, though the functional contribution of HCXmit was much higher in the liver than in the heart. Post-translational modifications or extra-mitochondrial localization of Letm1 protein in the heart, just as reported for NCLX [In the heart, the fraction of Nanditions ,23,24,25chondria ,27, was n et al.  detectedmit had been controversial [+ movements across the mitochondrial inner membrane via the electron transport chain, F1Fo-ATP synthase, and so on. Therefore, detecting an NCXmit-mediated ∆Ψ change with the exclusion of the ∆Ψ change via the fluctuation of energy metabolism was challenging. In order to overcome this problem, it is necessary to clamp ∆Ψ. Recently, our group succeeded in recording membrane currents through NCXmit in mouse cardiac mitochondria using whole-mitoplast patch clamp methods, thus settling the controversy [The electrogenicity of NCXoversial ,30,31,32troversy .mit current in forward mode, i.e., an extra-mitochondrial Na+-induced inward current with Ca2+ in the pipette, corresponded well to those in previous reports—the currents were diminished by the NCXmit inhibitor CGP-37157 [+ was around 3–4 [+ can be substitutable for Na+ with ~70% lower efficacy [+, 35.6 mM, was higher than the reported value of 1–8 mM in the heart  structure of NCLX has not been solved yet, recent advances in artificial intelligence-based structure prediction methods makes it possible to easily visualize a putative 3D structure of NCLX. The characteristics of the NCXGP-37157 ,33, the ound 3–4 ,32, and efficacy ,24. One  at mitochondria activated PKA, thereby phosphorylating S258 of NCLX . PINK1 d2+ handling, and so on [Mitochondria are physically and electrically connected with each other via intermitochondrial junctions and form “mitochondrial reticulum” throughout the cell in the skeletal and cardiac muscles ,46. Howend so on ,48.2+ indicator Myticam-expressing rabbit cardiomyocytes, Lu et al. [2+ uptake in IFM than in PNM, whereas the post-stimulation Ca2+ efflux was comparable. The higher uptake but comparable efflux of mitochondrial Ca2+ in IFM than in PNM resulted in the higher sensitivity to phenylarsine oxide for the opening of mPTP, shown as faster ∆Ψ depolarization. In addition, the spatial difference of CUmit activity was preserved in permeabilized cardiomyocytes under the conditions of a clamped cytosolic Ca2+ concentration with a disabled sarcoplasmic reticulum (SR) Ca2+ pump (SERCA), but the efficacy was lower than that observed in intact cardiomyocytes. Therefore, an intrinsic difference in CUmit activity may be further amplified by local SR Ca2+ release-associated excitation–contraction coupling in intact cardiomyocytes.Using mitochondrial Cau et al.  demonstr2+ efflux activity was comparable in IFM and PNM [+ channel Nav1.5 clusters, and hardly existed in IFM of rabbit ventricular myocytes. Functional analyses showed that the treatment of cells with a Na+ channel blocker, tetrodotoxin, increased intensity of Rhod-2, an indicator of mitochondrial Ca2+, in SSM but not in IFM. Since NCXmit inhibition by CGP-37157 produced similar results as those obtained using tetrodotoxin, it was suggested that Na+ accumulation at the subsarcolemmal space via Nav1.5 potentiated NCLX-mediated Ca2+ efflux from mitochondria, preventing mitochondrial Ca2+ accumulation. The authors further examined the contribution of the physical and functional coupling of Nav1.5-NCLX to reactive oxygen species (ROS) production in mitochondria, which is closely associated with mitochondrial Ca2+, as will be described in v1.5–NCLX coupling. Quantitative analysis of spatial Na+ regulation via the Na+-K+ ATPase and Na+ channel, as performed by Skogestad et al. [While Ca and PNM , it was  and PNM . Immunofd et al.  would he2+ dynamics at differentially localized cardiac mitochondria, intra-mitochondrial heterogeneities were also reported. Lu et al. [2+ transients using Myticam-expressing rat ventricular myocytes. They analyzed 0.2 Hz stimulation-induced small mitochondrial Ca2+ transients, which increased from ~150 nM by ~30 nM and found that the upstroke was faster at position near the Z-line than near the M-line but the decay was comparable. Although MCU immunofluorescence showed a uniform distribution over the mitochondrion, it was suggested that CUmit activity was higher at mitochondria facing junctional SR (jSR) than at those facing bulk cytosol, whereas Ca2+ efflux activity was comparable. This is reasonable because mitochondria–jSR association creates high Ca2+ microdomains near the dyadic space, which enables them to meet the low affinity of CUmit for Ca2+ uptake . 2+ dynamics. Using conventional and super-resolution immunofluorescence analyses of isolated cardiac mitochondria and isolated cardiomyocytes, they demonstrated that about 50% of MCU were closely co-localized with the SR Ca2+ release channel ryanodine receptor (RyR) 2 [mit regulator proteins, were more abundant in crude mitochondria than in Percoll-purified mitochondria, and were also found in jSR [mit activity—CUmit inhibitor Ru360-sensitive 45Ca2+ uptake corrected with citrate synthase activity—was much higher in isolated jSR than that in isolated mitochondria. On the contrary, 45Ca2+ retention assays revealed that Na+- and CGP-37157-sensitive mitochondrial Ca2+ efflux activity was much higher in pure mitochondria than that in jSR. This 45Ca2+ efflux activity became larger and smaller in heart-specific NCLX overexpressing and knockout mice, respectively. The authors proposed that the spatially separated distribution of MCU-RyR2 and NCLX contributes to minimizing the energy cost for maintaining ∆Ψ. In other word, if MCU-RyR2 were near NCLX, ∆Ψ would depolarize both due to Ca2+ influx via CUmit and due to Ca2+ efflux via NCXmit. Accordingly, the spatial separation of MCU-RyR2 and NCLX should be necessary for optimizing mitochondrial Ca2+ signals and energy cost. Interestingly, it was demonstrated that NCLX efficiently supplies Ca2+ from mitochondria to the SR/endoplasmic reticulum (ER) via SERCA, thereby regulating the automaticity of HL-1 cardiomyocytes, as well as antigen receptor-mediated Ca2+ signaling of B lymphocytes [2+ cycling between SR and mitochondria.De La Fuente et al. ,56 furth (RyR) 2 . The aut (RyR) 2  was attr (RyR) 2  gave nond in jSR . On the mit comprises only 1–2% of total Ca2+ removal from cardiomyocytes [mit to cytosolic Ca2+ transients and to action potential generation has been considered negligible. However, in a spontaneously beating cell line, HL-1, originating from mouse atrial myocytes, NCLX knockdown by siRNA significantly decelerated the upstrokes of action potentials and Ca2+ transients, and prolonged the cycle lengths [2+ level. Rather, it was demonstrated that NCLX knockdown decreased caffeine-responsive SR Ca2+ content and slowed subsequent SR Ca2+ reuptake rate, evaluated using a FRET protein Cameleon D1ER. Further analyses of a mathematical model combined with experimental validation revealed that (1) the automaticity of HL-1 cells was driven by the so-called “Ca2+ clock” mechanism, in which a Ca2+ leak from SR potentiates the inward current via sarcolemmal Na+-Ca2+ exchange to facilitate membrane depolarization, (2) NCLX knockdown decreased the Ca2+ supply from mitochondria to SR, thereby decelerating SR Ca2+ leak, delaying the activation of the inward current through sarcolemmal Na+-Ca2+ exchange, and thus delaying the activations of voltage-dependent Na+ and Ca2+ currents, causing a cycle length prolongation.Since CUmyocytes ,57,58, t lengths ,57,58, Nmit may be involved in abnormal automaticity of atria, such as atrial flutter and atrial ectopic tachycardia. In addition, it may also be plausible that abnormal NCXmit function causes ventricular arrhythmias. In fact, the involvement of abnormal NCXmit activity in altered rhythmicity was suggested in mouse embryonic stem cell-derived as well as in human induced pluripotent stem cell-derived ventricular myocytes, where the “Ca2+ clock” drives the automaticity [Considering that HL-1 cells are derived from atrial myocytes, which are quiescent under physiological conditions, NCXmaticity . In addimaticity .mit participates in the automaticity of normal pacemaker cells, i.e., sinoatrial (SA) node cells, is still a big issue. The automaticity of SA node cells has been proposed to be driven by a “coupled-clock” pacemaking system, which is composed of a sarcolemmal ion channel/transporter-derived rhythm (“membrane clock”) and subsarcolemmal Ca2+ release (LCR)-related rhythm (“Ca2+ clock”) [+-Ca2+ exchange to drive diastolic depolarization. NCXmit may modulate the “Ca2+ clock” part in SA node cells, as observed in HL-1 cells [mit inhibitor, CGP-37157, slowed the firing rate of rabbit as well as mouse SA node cells [2+ transients within and among SA node cells [2+ transients and did not generate LCR; and some generated LCR during the diastolic phase before an occurrence of action potential-induced Ca2+ transients. These data suggest that the coupling degree of the “coupled-clock” system may differ among SA node cells in vivo. Our model analyses suggested that NCXmit reduction in an SA node cell which is solely driven by the “membrane clock” accelerates, instead of decelerating, the firing rate [mit reduction-mediated slowing of automaticity in “Ca2+ clock”-driven cells may be compromised by NCXmit-mediated acceleration of automaticity in “membrane clock” cells in the SA node region. In fact, tamoxifen-induced NCLX deletion in the adult mouse heart, with a 70% reduction of NCLX protein 3 days after tamoxifen treatment, did not show altered sinus rhythms except for on the date of death, 8–10 days after tamoxifen treatment [mit in pacemaking activity.The question of whether NCX clock”) ,61,62. I-1 cells . In factde cells ,64. Howede cells . That ising rate . NCXmit reatment . In vivo2+, which activates three mitochondrial dehydrogenases—pyruvate dehydrogenase complex (PDHC), isocitrate dehydrogenase, and 2-oxoglutarate dehydrogenase (OGDH) [1Fo ATP synthase. Therefore, the contribution of NCXmit, as one of the determinants of the mitochondrial Ca2+ level, to cardiac energetics has been an issue to be clarified.The heart is continuously pumping blood around the body, which is energetically driven by ATP hydrolysis. In the healthy adult heart, ATP synthesis is mainly dependent on mitochondrial oxidative phosphorylation, and the process is strictly regulated to balance the large, dynamically changing energy demands . One cane (OGDH)   was diminished. At the same time, under the condition of 15 mM Na+ in the pipette, NADH autofluorescence decreased upon the workload increase, indicating that mitochondrial Ca2+ was not sufficient enough to activate NADH production by mitochondrial dehydrogenases. An NCXmit inhibitor, CGP-37157, restored the workload-induced Ca2+ accumulation in mitochondria and attenuated the NADH decrease [+ increase and energy starvation are characteristic properties of failing heart [+ evaluated from SBFI ratio image was ~15 mM compared to ~5 mM in sham myocytes, the abrupt workload increase caused essentially the same responses of mitochondrial Ca2+ (Rhod-2 or Myticam) and NADH as those reported with 15 mM Na+ in the pipette [2+ and the subsequent NADH starvation upon the workload increase. More importantly, the changes were restored in the presence of an NCXmit inhibitor, CGP-37157, to levels similar to those observed in sham myocytes. These results suggested a causative role of NCXmit in the energy starvation of the failing heart. In addition, as will be explained in mit is a novel strategy for treating heart failure [This was first shown in whole-cell patch clamp experiments using guinea pig ventricular myocytes loaded with Rhod-2 for evaluating mitochondrial Ca changes . It was decrease . Since ang heart , the autng heart ,71. In v pipette ,69—the dmit to cardiac energetics in the failing heart may not be as large as that expected from experiments using cardiomyocytes, where an extreme workload change was applied—rapid 3–4 Hz pacing from a quiescent state, which hearts in situ never experience [+ loads on cardiac energetics were extensively studied in Langendorff-perfused mouse hearts with 23Na, 31P, 13C NMR, and 1H-NMR metabolomic profiling [3SA mouse) and acute (treatment with ouabain and blebbistatin) inhibition of Na+-K+ ATPase, as well as pressure-overload-induced cardiac hypertrophy caused a cytosolic Na+ increase, and switched the substrate preference from fatty acid to carbohydrate oxidation, which are characteristic features frequently observed in failing hearts [+ elevation resulted in the most severe metabolic alterations, such as decreased metabolite levels of tricarboxylic acid (TCA) cycle intermediates downstream from OGDH , suggesting the reduced Ca2+-dependent activation of TCA cycle dehydrogenases. However, regardless of the strategy for cytosolic Na+ elevations, the energy supply was maintained, as is evident from the preserved ATP, phosphocreatine (PCr), PCr/ATP ratio, NADH, and pH. Metabolome profiles obtained with NMR, as well as in silico predictions using CardiNet, revealed that they were achieved at the cost of extensive metabolic flux remodeling. Therefore, the impact of impaired cytosolic Na+ homeostasis on mitochondrial ATP production should be mechanistically more complex than what has been suggested in isolated cardiomyocytes. In all three sets of hearts with elevated cytosolic Na+, treatment with CGP-37157 reversed the substrate preference from carbohydrate to fatty acid oxidation with normalized levels of the depleted metabolites. This suggests a therapeutic potential for CGP-37157 in the treatment of the metabolic reprograming that occurs before energetic impairments.However, the contribution of NCXperience ,69,71. Rrofiling . Chronicg hearts ,73. The mit to cardiac energetics were not suggested in heart-specific NCLX-overexpression mice [+/NADH ratio, oxidative phosphorylation evaluated by seahorse analyses with either pyruvate or palmitate as energy substrates , nor in the phosphorylation level of mitochondrial Ca2+-responsive PDHC. These findings suggested that NCLX overexpression had marginal effects on cardiac energetics. Rather, as will be explained in 2+ level, evaluated as carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone)-responsive Fura-2 intensity, was comparable between control and NCLX-overexpressing cardiomyocytes, indicating that NCLX overexpression did not cause excessive deprivation of mitochondrial Ca2+, in spite of an increase in mitochondrial Ca2+ efflux rate by 88%. Our model analyses suggested that cytosolic Ca2+ within its physiological range, 100 nM–2 μM, does not largely affect steady-state levels of energy substrates, though a lower cytosolic Ca2+ level collapsed the system because of mitochondrial Ca2+ deprivation [2+ deprivation via NCLX overexpression. It would be informative to further evaluate the mitochondrial Ca2+ level, cytosolic Na+ level, and metabolome profiles in failing hearts with or without NCLX overexpression.To the contrary, detrimental contributions of NCXion mice . There wrivation ,75. Ther2− from O2 oxidation. Then, manganese-dependent superoxide dismutase converts O2− to H2O2, which is eliminated by antioxidant scavenge systems, such as glutathione peroxidase and peroxiredoxin [2+. mPTP opening causes a burst of ROS released from mitochondria, in a process named ROS-induced ROS release, which impairs excitation–contraction coupling via modulating multiple ion channels and transporters, as well as via chronic remodeling [2+ activates three dehydrogenases and the product NADH promotes ROS by-production via the electron transport chain, and that mitochondrial Ca2+ overload is a key trigger for mPTP opening, followed by a burst ROS release, altered mitochondrial Ca2+ dynamics have been implicated to be closely associated with ROS dynamics in failing and injured hearts [Mitochondria are a major source of ROS, the production of which is tightly coupled with ATP synthesis—Complex I and Complex III in the electron transport chain produce Oiredoxin  H levels in failing cardiomyocytes. Accordingly, these findings indicated a detrimental contribution of NCXmit in failing hearts.However, a contradictory mechanism was proposed by Liu et al. . As expla NCXmit . Interesmit in failing hearts suggested that mitochondrial Ca2+ did not simply correlate with ROS production. Recently, a brand-new mechanism underlying hypoxia-induced ROS production via NCXmit was proposed—Na+–phospholipid interaction-mediated ROS regulation [mit was involved in hypoxia-induced ROS production in primary bovine aortic endothelial cells and mouse embryonic fibroblasts. Pharmacological inhibition with CGP-37157 or genetical reduction (siRNA or knockout) of NCLX diminished the cytosolic Ca2+ increase and cytosolic Na+ decrease, attenuated the reduction of the inner mitochondrial membrane fluidity and the mitochondrial ROS production caused by exposure of the cells to hypoxia (exposure of the cells to 1% O2). Then, the authors showed that hypoxia-induced matrix acidification via Complex I inhibition caused Ca2+ solubilization from calcium phosphate precipitation in the matrix, as evident from morphological (electron microscopy images) as well as from functional assays . Since a mitochondrial free Ca2+ increase enables NCXmit to extrude Ca2+ in exchange for Na+, the authors then focused on the roles of matrix Na+ on electron transport chains and found that only Complex II-dependent respirations were decreased by Na+, which was NCXmit-dependent, resulting in increased ROS production. The authors filled in the final piece by showing that Na+ directly bound to the phospholipid bilayer, as evident from infrared spectroscopy, which reduced the fluidity of the inner mitochondrial membrane for ubiquinone diffusion in the inner mitochondrial membrane, increasing the ROS production. Taken together, these findings clarified a distinct scheme of ROS production—regulation by matrix Na+ via NCXmit—from those proposed in previous reports.The abovementioned contradictory roles of NCXgulation   in which considerable cytosolic Na+ overload occurs, and the latter by heart failure with a preserved or moderate ejection fraction  in which only a modest Na+ increase is expected. Integrating the model of matrix Ca2+ solubilization and precipitation from and to calcium phosphate [In order to understand these complicated networks, the integration of NCXa et al.  succeede details ). In brihosphate  into Corhosphate  would fumit in healthy as well as in failing hearts will be further deepened.The discrepancies in experimental findings obtained from isolated mitochondria, isolated cardiomyocytes, and whole hearts are other issues that remain to be solved. Recent advances in imaging techniques used to evaluate electrophysiological and metabolic properties of single cells and even organelles in tissue are promising ,85. By u


Here, we investigated in computational analyses the properties of the permeation pathway of TPC1 from Arabidopsis thaliana. Using the crystal structure of AtTPC1, protein modeling, molecular dynamics (MD) simulations, and free energy calculations, we identified a free energy minimum for Ca2+, but not for K+, at the luminal side next to the selectivity filter. Residues D269 and E637 coordinate in particular Ca2+ as demonstrated in in silico mutagenesis experiments. Such a Ca2+-specific coordination site in the pore explains contradicting data for the relative Ca2+/K+ permeability and strongly suggests that the Ca2+ permeability of SV channels is largely overestimated from relative permeability analyses. This conclusion was further supported by in silico electrophysiological studies showing a remarkable permeation of K+ but not Ca2+ through the open channel.Two Pore Channels (TPCs) are cation-selective voltage- and ligand-gated ion channels in membranes of intracellular organelles of eukaryotic cells. In plants, the TPC1 subtype forms the slowly activating vacuolar (SV) channel, the most dominant ion channel in the vacuolar membrane. Controversial reports about the permeability properties of plant SV channels fueled speculations about the physiological roles of this channel type. TPC1 is thought to have high Ca For the model plant Arabidopsis thaliana, it was shown that the SV channel belongs to the class of Two Pore Channels (TPCs) and is encoded by the TPC1 gene [Shaker cassette structure that dimerizes via a C-terminal dimerization domain [Shaker cassette, the first four helices form a voltage-sensing domain (VSD), and helices 5 and 6 constitute the pore domain (P). The arrangement of the two subunits is domain-swapped, which means that the VSD1 of one Shaker cassette contacts P2 of the other cassette, while P1 contacts VSD2 of the same cassette. The four P-domains form the central permeation pathway.The Slowly activating Vacuolar (SV) channel was the first ion channel identified in plant vacuoles . It is an domain  to form n domain ,5. In ea2+ signaling and are essential players in vacuole excitability [+ and Na+ and divalent Ca2+ ions [Vicia faba, such analyses resulted in a permeability ratio of Ca:PKP ≈ 5:1 in one ionic condition [Ca:PKP ≈ 1:10 in another [2+ channel with a slight K+ permeability, but the second implies the opposite. This led to the ongoing discussion of whether a significant amount of Ca2+ can pass the TPC1/SV channels under physiological conditions. By combining the patch-clamp technique with fura-2 fluorescence detection, electrical currents and changes in the Ca2+ concentration could be measured in parallel [TPCs are ubiquitously found in organelles of animals and plants and exist in three types, TPC1, 2, and 3, each with different gating and ion selectivity. They are ubiquitously found in organelles of animals and plants ,7. Nevertability ,11,12. Pa2+ ions ,26,27,28a2+ ions , and perondition  and PCa: another . The firparallel . This apTo shed light on this puzzling set of partially contradicting reports about the permeability properties of TPC1, here we approached this topic from a structural and computational point of view. Using protein modeling, molecular dynamics (MD) simulations, and free energy calculations, we investigated the permeation patterns of different ions in AtTPC1 to elucidate which residues are involved in the translocation of ions and understand which structural features determine the selectivity of AtTPC1.sn-glycero-3-phosphocholine (POPC). We established three different systems with salt concentrations of 0.15 M NaCl, 0.15 M KCl, or 0.1 M CaCl2. The systems were allowed to relax in a series of energy minimizations and heating simulations. Subsequently, 250 ns of molecular dynamics simulations were carried out for five independent replicas for each saline concentration. The pore and, in particular, the selectivity filter (SF) of AtTPC1 became markedly narrower in the presence of Na+ and Ca2+ compared to the system with K+  and embedded it in an explicit membrane of 1-palmitoyl-2-oleoyl- with K+ . Hence, +, K+, or Ca2+) was selected to go through the channel pore. As a negative control, Cl− was taken from the KCl condition in an independent run. SMD simulations were carried out in two stages. First, the ion to be analyzed was aligned with the center of the SF at the luminal side in parallel to the membrane  simulations followed by umbrella sampling (US) simulations. For each salt condition, a single cation , the largest free energy barrier was 2.8 kcal·mol−1 for Ca2+, followed by 2.1 kcal·mol−1 for Na+, and 0.9 kcal·mol−1 for K+. Additionally, the energetic analyses pointed to a minimum in the PMF of −4.1 kcal·mol−1 for Ca2+ at the luminal side before the SF. Therefore, a Ca2+ ion permeating from the vacuolar lumen to the cytosol had to overcome an energy barrier of ~6.9 kcal·mol−1, whereas the largest barrier for a K+ ion  was only ~20% of this value. The largest barrier for Na+  was about twice as high as the barrier for K+. Besides the PMF, the US simulations allowed us to estimate the number of water molecules surrounding the investigated ions  simulations, the potentials of mean force (PMF) for the different ions at the remaining 70 positions along the pore were computed. The PMFs indicated significant differences between Cland Ca2+ B,C. Aparted ions D. At varure data . It has  than K+ ,35 and tn for K+ . Conside2+ affinity in the AtTPC1 pore, we inspected the region at the luminal side before the SF. We identified four negatively charged residues . Each system was equilibrated in MD simulations of 250 ns length.For a more in-depth investigation of the ion permeation mechanism through AtTPC1, we constructed a homology model of this channel in the open configuration. We followed the protocol established by Guo et al.  to generE = +0.180 kcal mol−1 Å−1 e−1 or E = −0.180 kcal mol−1 Å−1 e−1 were applied along the permeation pathway in the direction of the z-axis [t = 500 ns. For all six conditions, K+/+ΔV, K+/−ΔV, Na+/+ΔV, Na+/−ΔV, Ca2+/+ΔV, and Ca2+/−ΔV, we ran five independent simulations. In the time interval of Δt = 500 ns, we counted from 0 to 100 K+ ions , we counted 34 ± 39 K+ ions, while we registered 24 ± 22 K+ ions in the inverse direction from the lumen to the cytosol (−ΔV). When we repeated the forced ion flux simulations with the mutant AtTPC1-D269A-E637A, we observed that this mutation strongly reduced ion mobility for all three ions  and S1/SF closer to the luminal side, and S2  and Scyt (D306 and Y305) closer to the cytosolic side  as well as polar interactions (S1/SF and S2) dictated the passage of ions through the channel. When comparing +ΔV (K+ efflux) and −ΔV (K+ influx) simulations, we did not observe structural differences at positions Scyt, S2, and S1/SF. By contrast, two different configurations of Svac were observed , we observed that the D605/D606/E607 motif moved away from the Svac site  fit well into the AtTPC1 pore, we were unable to provide substantial evidence that Ca2+ ions can permeate the channel better than K+. On the contrary, the pore seems to have a predisposition for K+ over Ca2+. This result appears to be contradictory to several experimental findings. AtTPC1 has been assigned a relative permeability sequence of Ca2+ > Na+∼Li+∼K+ > Rb+ > Cs+ [2+ is difficult to handle in molecular simulations [+ and Ca2+, for instance, necessarily interact with each other  at the cytosolic side and 15 mM Ca2+ at the luminal side. At 0 mV, the current is zero, which means that under this condition, the inwardly directed chemical Ca2+ gradient equals the outwardly directed K+ gradient (+ and Ca2+ do not interact with each other (prerequisites of the GHK equation), this would indeed mean that Ca2+ permeates better than K+. In such a condition, a ten-fold lower Ca2+ gradient would be sufficient to balance the flux induced by the K+ gradient. From the crystal structure and our MD simulations we conclude, however, that the permeation process through AtTPC1 is fundamentally different from that of the GHK model. When Ca2+ moves from the lumen to the cytosol and K+ from the cytosol to the lumen, both ions need to use the same pathway. In such a scenario, zero current means essentially no net flux at all (2+ in one direction is compensated by the charge flux carried by K+ in the other (2+ and K+ block each other. The reason why just one tenth of the Ca2+ concentration is sufficient to block the K+ (Na+) flux at 0 mV [2+ coordination site in plant TPC1s on the luminal side next to the selectivity filter. The identified site might not only have an influence on ion permeation but could also be responsible for ion concentration-dependent changes in the gating properties of SV channels [2+ ion at that position, which effectively has a concentrating effect. The vacuolar Ca2+ concentration used in the GHK equation is therefore a dramatic underestimate of the local surface Ca2+ concentration at the luminal entrance of the permeation pathway.The obtained “relative permeabilities” could therefore be strongly misleading, such as a Por TPC1s ,39. Suchgradient A. If thex at all B, while he other A. In othchannels . The freCa/PK > 1 even though Ca2+ permeates less favorably the AtTPC1 pore. This finding fits with the experimental observation of TPC1s having a higher conductance for K+/Na+ than for Ca2+ [+ permeates markedly better than Ca2+ . The structure was prepared using the PRIME module of the Schrodinger suite of programs .sn-glycero-3-phosphocholine (POPC) using the PACKMOL-Memgen module [2 for each protein system using the TIP3P water model [We embedded AtTPC1 in an explicit membrane of 1-palmitoyl-2-oleoyl-n module  of the An module ,48. To aer model . The gener model ,50 with er model , includier model  were app+, K+, or Ca2+) was selected to go through the channel pore. Additionally, Cl− was taken from the KCl condition as a negative control in an independent run. Two runs of SMD simulations were performed. In the first run, a force with a constant of 1.0 kcal·mol−1·Å−2 was applied to the ion for 0.01 ns, aligning it with the center of mass (COM) of the SF of AtTPC1 in the xy plane (membrane plane), while maintaining a distance of ~26 Å in the z-axis, which is perpendicular to the membrane plane. To run multiple single axis restraints, sander was used. In the following, a second pulling simulation was performed, applying a force with a constant of 1.0 kcal·mol−1·Å−2 for the monovalent ions and 1.5 kcal·mol−1·Å−2 for the divalent ions, to move the ion across the protein normal to the membrane plane, placing it at a distance of ~50 Å from the starting point below the SF on the cytosolic side during 0.130 ns. From the latter simulation, 100 frames in which the ion was at a distance of 0.5 Å with respect to each other were selected as starting conformations for the US simulations. During the US simulations, the distance to the SF in the z-axis was used as a reaction coordinate, using a harmonic potential with a force constant of 10 kcal·mol−1·Å−2 to maintain the ion position. In order to avoid lateral movements of the ion and to ensure a proper sampling in the space surrounding the pore of the channels, we used a flat-bottom restraint in the xy-plane with radius 8 Å with respect to the COM of the SF and a harmonic restraint with a force constant of 10 kcal·mol−1·Å−2. To prevent other ions from accessing the SF, two distance restraints with respect to the center of mass of the SF and to the residues forming the cross region were applied. For both, a force constant of 5 kcal·mol−1·Å−2 was used and a minimum distance of 15 Å from each reference point was enforced. For each window, 10 ns of MD simulation were performed, from which the first 3 ns were discarded as equilibration, and the remaining 7 ns were used in order to generate the PMF. To estimate the unbiased free energy profile for the ion permeation, the WHAM algorithm was used [−7. In each PMF, the first and last 10 points are considered in the bulk water and used to shift the profile. The error was estimated from the PMF of every nanosecond of the production runs. The width of the permeation pathway was monitored with the program HOLE [To properly analyze the differences in affinity between the tested ions and AtTPC1, Steered Molecular Dynamics (SMD) simulations followed by Umbrella Sampling (US) simulations were performed. For each salt condition, a single cation (Nawas used  with a tram HOLE .2). Subsequently, external fields with intensities of E = ±0.180 kcal·mol−1·Å−1·e−1 were applied in the direction of the z-axis to induce inward and outward currents, respectively. All ion permeations events were measured by an in-house script.To investigate the ion permeation mechanism through TPC, an open configuration of AtTPC1 was constructed. A homology model was built using the crystal structure of an open NavMs sodium channel PDB ID 5hvx) as an additional template. We followed the strategy described by Guo et al. hvx as an, we crea