Structural architecture of the human NALCN channelosome.

Depolarizing sodium (Na+) leak currents carried by the NALCN channel regulate the resting membrane potential of many neurons to modulate respiration, circadian rhythm, locomotion and pain sensitivity1-8. NALCN requires FAM155A, UNC79 and UNC80 to function, but the role of these auxiliary subunits is not understood3,7,9-12. NALCN, UNC79 and UNC80 are essential in rodents2,9,13, and mutations in human NALCN and UNC80 cause severe developmental and neurological disease14,15. Here we determined the structure of the NALCN channelosome, an approximately 1-MDa complex, as fundamental aspects about the composition, assembly and gating of this channelosome remain obscure. UNC79 and UNC80 are massive HEAT-repeat proteins that form an intertwined anti-parallel superhelical assembly, which docks intracellularly onto the NALCN-FAM155A pore-forming subcomplex. Calmodulin copurifies bound to the carboxy-terminal domain of NALCN, identifying this region as a putative modulatory hub. Single-channel analyses uncovered a low open probability for the wild-type complex, highlighting the tightly closed S6 gate in the structure, and providing a basis to interpret the altered gating properties of disease-causing variants. Key constraints between the UNC79-UNC80 subcomplex and the NALCN DI-DII and DII-DIII linkers were identified, leading to a model of channelosome gating. Our results provide a structural blueprint to understand the physiology of the NALCN channelosome and a template for drug discovery to modulate the resting membrane potential.

NEDD4L intramolecular interactions regulate its auto and substrate NaV1.5 ubiquitination.

NEDD4L is a HECT-type E3 ligase that catalyzes the addition of ubiquitin to intracellular substrates such as the cardiac voltage-gated sodium channel, NaV1.5. The intramolecular interactions of NEDD4L regulate its enzymatic activity which is essential for proteostasis. For NaV1.5, this process is critical as alterations in Na+ current is involved in cardiac diseases including arrhythmias and heart failure. In this study, we perform extensive biochemical and functional analyses that implicate the C2 domain and the first WW-linker (1,2-linker) in the autoregulatory mechanism of NEDD4L. Through in vitro and electrophysiological experiments, the NEDD4L 1,2-linker was determined to be important in substrate ubiquitination of NaV1.5. We establish the preferred sites of ubiquitination of NEDD4L to be in the second WW-linker (2,3-linker). Interestingly, NEDD4L ubiquitinates the cytoplasmic linker between the first and second transmembrane domains of the channel (DI-DII) of NaV1.5. Moreover, we design a genetically encoded modulator of Nav1.5 that achieves Na+ current reduction using the NEDD4L HECT domain as cargo of a NaV1.5-binding nanobody. These investigations elucidate the mechanisms regulating the NEDD4 family and furnish a new molecular framework for understanding NaV1.5 ubiquitination.

Elementary mechanisms of calmodulin regulation of NaV1.5 producing divergent arrhythmogenic phenotypes.

In cardiomyocytes, NaV1.5 channels mediate initiation and fast propagation of action potentials. The Ca2+-binding protein calmodulin (CaM) serves as a de facto subunit of NaV1.5. Genetic studies and atomic structures suggest that this interaction is pathophysiologically critical, as human mutations within the NaV1.5 carboxy-terminus that disrupt CaM binding are linked to distinct forms of life-threatening arrhythmias, including long QT syndrome 3, a "gain-of-function" defect, and Brugada syndrome, a "loss-of-function" phenotype. Yet, how a common disruption in CaM binding engenders divergent effects on NaV1.5 gating is not fully understood, though vital for elucidating arrhythmogenic mechanisms and for developing new therapies. Here, using extensive single-channel analysis, we find that the disruption of Ca2+-free CaM preassociation with NaV1.5 exerts two disparate effects: 1) a decrease in the peak open probability and 2) an increase in persistent NaV openings. Mechanistically, these effects arise from a CaM-dependent switch in the NaV inactivation mechanism. Specifically, CaM-bound channels preferentially inactivate from the open state, while those devoid of CaM exhibit enhanced closed-state inactivation. Further enriching this scheme, for certain mutant NaV1.5, local Ca2+ fluctuations elicit a rapid recruitment of CaM that reverses the increase in persistent Na current, a factor that may promote beat-to-beat variability in late Na current. In all, these findings identify the elementary mechanism of CaM regulation of NaV1.5 and, in so doing, unravel a noncanonical role for CaM in tuning ion channel gating. Furthermore, our results furnish an in-depth molecular framework for understanding complex arrhythmogenic phenotypes of NaV1.5 channelopathies.

CaV channels reject signaling from a second CaM in eliciting Ca2+-dependent feedback regulation.

Calmodulin (CaM) regulation of voltage-gated calcium (CaV1-2) channels is a powerful Ca2+-feedback mechanism to adjust channel activity in response to Ca2+ influx. Despite progress in resolving mechanisms of CaM-CaV feedback, the stoichiometry of CaM interaction with CaV channels remains ambiguous. Functional studies that tethered CaM to CaV1.2 suggested that a single CaM sufficed for Ca2+ feedback, yet biochemical, FRET, and structural studies showed that multiple CaM molecules interact with distinct interfaces within channel cytosolic segments, suggesting that functional Ca2+ regulation may be more nuanced. Resolving this ambiguity is critical as CaM is enriched in subcellular domains where CaV channels reside, such as the cardiac dyad. We here localized multiple CaMs to the CaV nanodomain by tethering either WT or mutant CaM that lack Ca2+-binding capacity to the pore-forming α-subunit of CaV1.2, CaV1.3, and CaV2.1 and/or the auxiliary ß2A subunit. We observed that a single CaM tethered to either the α or ß2A subunit tunes Ca2+ regulation of CaV channels. However, when multiple CaMs are localized concurrently, CaV channels preferentially respond to signaling from the α-subunit-tethered CaM. Mechanistically, the introduction of a second IQ domain to the CaV1.3 carboxyl tail switched the apparent functional stoichiometry, permitting two CaMs to mediate functional regulation. In all, Ca2+ feedback of CaV channels depends exquisitely on a single CaM preassociated with the α-subunit carboxyl tail. Additional CaMs that colocalize with the channel complex are unable to trigger Ca2+-dependent feedback of channel gating but may support alternate regulatory functions.

The TRPM4 channel is functionally important for the beneficial cardiac remodeling induced by endurance training.

Cardiac hypertrophy (CH) is an adaptive process that exists in two distinct forms and allows the heart to adequately respond to an organism's needs. The first form of CH is physiological, adaptive and reversible. The second is pathological, irreversible and associated with fibrosis and cardiomyocyte death. CH involves multiple molecular mechanisms that are still not completely defined but it is now accepted that physiological CH is associated more with the PI3-K/Akt pathway while the main signaling cascade activated in pathological CH involves the Calcineurin-NFAT pathway. It was recently demonstrated that the TRPM4 channel may act as a negative regulator of pathological CH by regulating calcium entry and thus the Cn-NFAT pathway. In this study, we examined if the TRPM4 channel is involved in the physiological CH process. We evaluated the effects of 4 weeks endurance training on the hearts of Trpm4 +/+ and Trpm4 -/- mice. We identified an elevated functional expression of the TRPM4 channel in cardiomyocytes after endurance training suggesting a potential role for the channel in physiological CH. We then observed that Trpm4 +/+ mice displayed left ventricular hypertrophy after endurance training associated with enhanced cardiac function. By contrast, Trpm4 -/- mice did not develop these adaptions. While Trpm4 -/- mice did not develop gross cardiac hypertrophy, the cardiomyocyte surface area was larger and associated with an increase of Tunel positive cells. Endurance training in Trpm4 +/+ mice did not increase DNA fragmentation in the heart. Endurance training in Trpm4 +/+ mice was associated with activation of the classical physiological CH Akt pathway while Trpm4 -/- favored the Calcineurin pathway. Calcium studies demonstrated that TRPM4 channel negatively regulates calcium entry providing support for activation of the Cn-NFAT pathway in Trpm4 -/- mice. In conclusion, we provide evidence for the functional expression of TRPM4 channel in response to endurance training. This expression may help to maintain the balance between physiological and pathological hypertrophy.

Fibroblast growth factor homologous factors serve as a molecular rheostat in tuning arrhythmogenic cardiac late sodium current.

Voltage-gated sodium (Nav1.5) channels support the genesis and brisk spatial propagation of action potentials in the heart. Disruption of NaV1.5 inactivation results in a small persistent Na influx known as late Na current (I Na,L), which has emerged as a common pathogenic mechanism in both congenital and acquired cardiac arrhythmogenic syndromes. Here, using low-noise multi-channel recordings in heterologous systems, LQTS3 patient-derived iPSCs cardiomyocytes, and mouse ventricular myocytes, we demonstrate that the intracellular fibroblast growth factor homologous factors (FHF1-4) tune pathogenic I Na,L in an isoform-specific manner. This scheme suggests a complex orchestration of I Na,L in cardiomyocytes that may contribute to variable disease expressivity of NaV1.5 channelopathies. We further leverage these observations to engineer a peptide-inhibitor of I Na,L with a higher efficacy as compared to a well-established small-molecule inhibitor. Overall, these findings lend insights into molecular mechanisms underlying FHF regulation of I Na,L in pathophysiology and outline potential therapeutic avenues.

Probing ion channel macromolecular interactions using fluorescence resonance energy transfer.

Ion channels are macromolecular complexes whose functions are exquisitely tuned by interacting proteins. Fluorescence resonance energy transfer (FRET) is a powerful methodology that is adept at quantifying ion channel protein-protein interactions in living cells. For FRET experiments, the interacting partners are tagged with appropriate donor and acceptor fluorescent proteins. If the fluorescently-labeled molecules are in close proximity, then photoexcitation of the donor results in non-radiative energy transfer to the acceptor, and subsequent fluorescence emission of the acceptor. The stoichiometry of ion channel interactions and their relative binding affinities can be deduced by quantifying both the FRET efficiency and the total number of donors and acceptors in a given cell. In this chapter, we discuss general considerations for FRET analysis of biological interactions, various strategies for estimating FRET efficiencies, and detailed protocols for construction of binding curves and determination of stoichiometry. We focus on implementation of FRET assays using a flow cytometer given its amenability for high-throughput data acquisition, enhanced accessibility, and robust analysis. This versatile methodology permits mechanistic dissection of dynamic changes in ion channel interactions.

New role of TRPM4 channel in the cardiac excitation-contraction coupling in response to physiological and pathological hypertrophy in mouse.

The transient receptor potential Melastatin 4 (TRPM4) channel is a calcium-activated non-selective cation channel expressed widely. In the heart, using a knock-out mouse model, the TRPM4 channel has been shown to be involved in multiple processes, including ß-adrenergic regulation, cardiac conduction, action potential duration and hypertrophic adaptations. This channel was recently shown to be involved in stress-induced cardiac arrhythmias in a mouse model overexpressing TRPM4 in ventricular cardiomyocytes. However, the link between TRPM4 channel expression in ventricular cardiomyocytes, the hypertrophic response to stress and/or cellular arrhythmias has yet to be elucidated. In this present study, we induced pathological hypertrophy in response to myocardial infarction using a mouse model of Trpm4 gene invalidation, and demonstrate that TRPM4 is essential for survival. We also demonstrate that the TRPM4 is required to activate both the Akt and Calcineurin pathways. Finally, using two hypertrophy models, either a physiological response to endurance training or a pathological response to myocardial infarction, we show that TRPM4 plays a role in regulating transient calcium amplitudes and leads to the development of cellular arrhythmias potentially in cooperation with the Sodium-calcium exchange (NCX). Here, we report two functions of the TRPM4 channel: first its role in adaptive hypertrophy, and second its association with NCX could mediate transient calcium amplitudes which trigger cellular arrhythmias.

Screening for in-vivo regional contractile defaults to predict the delayed Doxorubicin Cardiotoxicity in Juvenile Rat.

Anthracyclines are key chemotherapeutic agents used in various adult and pediatric cancers, however, their clinical use is limited due to possible congestive heart failure (HF) caused by acute and irreversible cardiotoxicity. Currently, there is no method to predict the future development of the HF in these patients. In order to identify early biomarkers to predict anthracycline cardiotoxicity in long-term survivors of childhood cancer, this longitudinal study aimed to analyze early and late in-vivo regional myocardial anthracycline-induced cardiotoxicity, related to in-vitro cardiac myocytes dysfunction, in a juvenile rat model. Methods: Young male Wistar rats (4 weeks-old) were treated with different cumulative doses of doxorubicin (7.5, 10 or 12.5 mg/kg) or NaCl (0.9%) once a week for 6 weeks by intravenous injection. Cardiac function was evaluated in-vivo by conventional (left ventricular ejection fraction, LVEF) and regional two-dimensional (2D) speckle tracking echocardiography over the 4 months after the last injection. The animals were assigned to preserved (pEF) or reduced EF (rEF) groups at the end of the protocol and were compared to controls. Results: We observed a preferential contractile dysfunction of the base of the heart, further altered in the posterior segment, even in pEF group. The first regional alterations appeared 1 month after chemotherapy. Functional investigation of cardiomyocytes isolated from the LV base 1 month after doxorubicin treatment showed that early in-vivo contractile alterations were associated with both decreased myofilament Ca2+ sensitivity and length-dependent activation. Changes in post-translational modifications (phosphorylation; S-glutathionylation) and protein degradation of the cardiac myosin binding protein-C may contribute to these alterations. Conclusion: Our data suggest that screening of the contractile defaults of the base of the heart by regional 2D strain echocardiography is useful to detect subclinical myocardial dysfunction prior to the development of delayed anthracycline-induced cardiomyopathy in pediatric onco-cardiology.

Fibroblast growth factor homologous factors tune arrhythmogenic late NaV1.5 current in calmodulin binding-deficient channels.

The Ca2+-binding protein calmodulin has emerged as a pivotal player in tuning Na+ channel function, although its impact in vivo remains to be resolved. Here, we identify the role of calmodulin and the NaV1.5 interactome in regulating late Na+ current in cardiomyocytes. We created transgenic mice with cardiac-specific expression of human NaV1.5 channels with alanine substitutions for the IQ motif (IQ/AA). The mutations rendered the channels incapable of binding calmodulin to the C-terminus. The IQ/AA transgenic mice exhibited normal ventricular repolarization without arrhythmias and an absence of increased late Na+ current. In comparison, transgenic mice expressing a lidocaine-resistant (F1759A) human NaV1.5 demonstrated increased late Na+ current and prolonged repolarization in cardiomyocytes, with spontaneous arrhythmias. To determine regulatory factors that prevent late Na+ current for the IQ/AA mutant channel, we considered fibroblast growth factor homologous factors (FHFs), which are within the NaV1.5 proteomic subdomain shown by proximity labeling in transgenic mice expressing NaV1.5 conjugated to ascorbate peroxidase. We found that FGF13 diminished late current of the IQ/AA but not F1759A mutant cardiomyocytes, suggesting that endogenous FHFs may serve to prevent late Na+ current in mouse cardiomyocytes. Leveraging endogenous mechanisms may furnish an alternative avenue for developing novel pharmacology that selectively blunts late Na+ current.

Stress-induced protein S-glutathionylation and phosphorylation crosstalk in cardiac sarcomeric proteins - Impact on heart function.

BACKGROUND: The interplay between oxidative stress and other signaling pathways in the contractile machinery regulation during cardiac stress and its consequences on cardiac function remains poorly understood. We evaluated the effect of the crosstalk between ß-adrenergic and redox signaling on post-translational modifications of sarcomeric regulatory proteins, Myosin Binding Protein-C (MyBP-C) and Troponin I (TnI). METHODS AND RESULTS: We mimicked in vitro high level of physiological cardiac stress by forcing rat hearts to produce high levels of oxidized glutathione. This led to MyBP-C S-glutathionylation associated with lower protein kinase A (PKA) dependent phosphorylations of MyBP-C and TnI, increased myofilament Ca2+ sensitivity, and decreased systolic and diastolic properties of the isolated perfused heart. Moderate physiological cardiac stress achieved in vivo with a single 35 min exercise (Low stress induced by exercise, LSE) increased TnI and cMyBP-C phosphorylations and improved cardiac function in vivo (echocardiography) and ex-vivo (isolated perfused heart). High stress induced by exercise (HSE) altered strongly oxidative stress markers and phosphorylations were unchanged despite increased PKA activity. HSE led to in vivo intrinsic cardiac dysfunction associated with myofilament Ca2+ sensitivity defects. To limit protein S-glutathionylation after HSE, we treated rats with N-acetylcysteine (NAC). NAC restored the ability of PKA to modulate myofilament Ca2+ sensitivity and prevented cardiac dysfunction observed in HSE animals. CONCLUSION: Under cardiac stress, adrenergic and oxidative signaling pathways work in concert to alter myofilament properties and are key regulators of cardiac function.