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Voltage-gated sodium (Nav1.5) channels support the genesis and brisk spatial propagation of action potentials in the heart. Disruption of Na V 1.5 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 Na V 1.5 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., Competing Interests: Competing Interests N.C., S.O.M, and M.B.-J., (inventors) filed a provisional patent (attorney docket no. CoU1046P/CU22077; filed 14 February 2022) for application of FixR for inhibiting late Na current. The remaining authors declare no competing interests.

Neuronal voltage-gated sodium channel Na V 1.2 C-terminal domain (CTD) binds calmodulin (CaM) constitutively at its IQ motif. A solution structure (6BUT) and other NMR evidence showed that the CaM N domain (CaM N ) is structurally independent of the C-domain (CaM C ) whether CaM is bound to the Na V 1.2 IQp (1,901-1,927) or Na V 1.2 CTD (1,777-1,937) with or without calcium. However, in the CaM + Na V 1.2 CTD complex, the Ca 2+ affinity of CaM N was more favorable than in free CaM, while Ca 2+ affinity for CaM C was weaker than in the CaM + Na V 1.2 IQp complex. The CTD EF-like (EFL) domain allosterically widened the energetic gap between CaM domains. Cardiomyopathy-associated CaM mutants (N53I(N54I), D95V(D96V), A102V(A103V), E104A(E105A), D129G(D130G), and F141L(F142L)) all bound the Na V 1.2 IQ motif favorably under resting (apo) conditions and bound calcium normally at CaM N sites. However, only N53I and A102V bound calcium at CaM C sites at [Ca 2+ ] < 100 μM. Thus, they are expected to respond like wild-type CaM to Ca 2+ spikes in excitable cells., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Voltage-gated sodium channels (Na v s) are tightly regulated by multiple conserved auxiliary proteins, including the four fibroblast growth factor homologous factors (FGFs), which bind the Na v EF-hand like domain (EFL), and calmodulin (CaM), a multifunctional messenger protein that binds the Na V IQ motif. The EFL domain and IQ motif are contiguous regions of Na V cytosolic C-terminal domains (CTD), placing CaM and FGF in close proximity. However, whether the FGFs and CaM act independently, directly associate, or operate through allosteric interactions to regulate channel function is unknown. Titrations monitored by steady-state fluorescence spectroscopy, structural studies with solution NMR, and computational modeling demonstrated for the first time that both domains of (Ca 2+ ) 4 -CaM (but not apo CaM) directly bind two sites in the N-terminal domain (NTD) of A-type FGF splice variants (FGF11A, FGF12A, FGF13A, and FGF14A) with high affinity. The weaker of the (Ca 2+ ) 4 -CaM-binding sites was known via electrophysiology to have a role in long-term inactivation of the channel but not known to bind CaM. FGF12A binding to a complex of CaM associated with a fragment of the Na V 1.2 CTD increased the Ca 2+ -binding affinity of both CaM domains, consistent with (Ca 2+ ) 4 -CaM interacting preferentially with its higher-affinity site in the FGF12A NTD. Thus, A-type FGFs can compete with Na V IQ motifs for (Ca 2+ ) 4 -CaM. During spikes in the cytosolic Ca 2+ concentration that accompany an action potential, CaM may translocate from the Na V IQ motif to the FGF NTD, or the A-type FGF NTD may recruit a second molecule of CaM to the channel., Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Human voltage-gated sodium (Na V ) channels are critical for initiating and propagating action potentials in excitable cells. Nine isoforms have different roles but similar topologies, with a pore-forming α-subunit and auxiliary transmembrane β-subunits. Na V pathologies lead to debilitating conditions including epilepsy, chronic pain, cardiac arrhythmias, and skeletal muscle paralysis. The ubiquitous calcium sensor calmodulin (CaM) binds to an IQ motif in the C-terminal tail of the α-subunit of all Na V isoforms, and contributes to calcium-dependent pore-gating in some channels. Previous structural studies of calcium-free (apo) CaM bound to the IQ motifs of Na V 1.2, Na V 1.5, and Na V 1.6 showed that CaM binding was mediated by the C-domain of CaM (CaM C ), while the N-domain (CaM N ) made no detectable contacts. To determine whether this domain-specific recognition mechanism is conserved in other Na V isoforms, we used solution NMR spectroscopy to assign the backbone resonances of complexes of apo CaM bound to peptides of IQ motifs of Na V 1.1, Na V 1.4, and Na V 1.7. Analysis of chemical shift differences showed that peptide binding only perturbed resonances in CaM C ; resonances of CaM N were identical to free CaM. Thus, CaM C residues contribute to the interface with the IQ motif, while CaM N is available to interact elsewhere on the channel.

Human voltage-gated sodium channel Na V 1.2 has a single pore-forming α-subunit and two transmembrane β-subunits. Expressed primarily in the brain, Na V 1.2 is critical for initiation and propagation of action potentials. Milliseconds after the pore opens, sodium influx is terminated by inactivation processes mediated by regulatory proteins including calmodulin (CaM). Both calcium-free (apo) CaM and calcium-saturated CaM bind tightly to an IQ motif in the C-terminal tail of the α-subunit. Our thermodynamic studies and solution structure (2KXW) of a C-domain fragment of apo 13 C, 15 N- CaM (CaM C ) bound to an unlabeled peptide with the sequence of rat Na V 1.2 IQ motif showed that apo CaM C (a) was necessary and sufficient for binding, and (b) bound more favorably than calcium-saturated CaM C . However, we could not monitor the Na V 1.2 residues directly, and no structure of full-length CaM (including the N-domain of CaM (CaM N )) was determined. To distinguish contributions of CaM N and CaM C , we used solution NMR spectroscopy to assign the backbone resonances of a complex containing a 13 C, 15 N-labeled peptide with the sequence of human Na V 1.2 IQ motif (Na V 1.2 IQp ) bound to apo 13 C, 15 N-CaM or apo 13 C, 15 N-CaM C . Comparing the assignments of apo CaM in complex with Na V 1.2 IQp to those of free apo CaM showed that residues within CaM C were significantly perturbed, while residues within CaM N were essentially unchanged. The chemical shifts of residues in Na V 1.2 IQp and in the C-domain of CaM were nearly identical regardless of whether CaM N was covalently linked to CaM C . This suggests that CaM N does not influence apo CaM binding to Na V 1.2 IQp .

Glucocorticoids (GCs), including dexamethasone (dex), are a central component of combination chemotherapy for childhood B-cell precursor acute lymphoblastic leukemia (B-ALL). GCs work by activating the GC receptor (GR), a ligand-induced transcription factor, which in turn regulates genes that induce leukemic cell death. Which GR-regulated genes are required for GC cytotoxicity, which pathways affect their regulation, and how resistance arises are not well understood. Here, we systematically integrate the transcriptional response of B-ALL to GCs with a next-generation short hairpin RNA screen to identify GC-regulated "effector" genes that contribute to cell death, as well as genes that affect the sensitivity of B-ALL cells to dex. This analysis reveals a pervasive role for GCs in suppression of B-cell development genes that is linked to therapeutic response. Inhibition of phosphatidylinositol 3-kinase δ (PI3Kδ), a linchpin in the pre-B-cell receptor and interleukin 7 receptor signaling pathways critical to B-cell development (with CAL-101 [idelalisib]), interrupts a double-negative feedback loop, enhancing GC-regulated transcription to synergistically kill even highly resistant B-ALL with diverse genetic backgrounds. This work not only identifies numerous opportunities for enhanced lymphoid-specific combination chemotherapies that have the potential to overcome treatment resistance, but is also a valuable resource for understanding GC biology and the mechanistic details of GR-regulated transcription., (© 2017 by The American Society of Hematology.)

Several members of the voltage-gated sodium channel family are regulated by calmodulin (CaM) and ionic calcium. The neuronal voltage-gated sodium channel Na V 1.2 contains binding sites for both apo (calcium-depleted) and calcium-saturated CaM. We have determined equilibrium dissociation constants for rat Na V 1.2 IQ motif [IQRAYRRYLLK] binding to apo CaM (~3nM) and (Ca 2+ ) 4 -CaM (~85nM), showing that apo CaM binding is favored by 30-fold. For both apo and (Ca 2+ ) 4 -CaM, NMR demonstrated that Na V 1.2 IQ motif peptide (Na V 1.2 IQp ) exclusively made contacts with C-domain residues of CaM (CaM C ). To understand how calcium triggers conformational change at the CaM-IQ interface, we determined a solution structure (2M5E.pdb) of (Ca 2+ ) 2 -CaM C bound to Na V 1.2 IQp . The polarity of (Ca 2+ ) 2 -CaM C relative to the IQ motif was opposite to that seen in apo CaM C -Na v 1.2 IQp (2KXW), revealing that CaM C recognizes nested, anti-parallel sites in Na v 1.2 IQp . Reversal of CaM may require transient release from the IQ motif during calcium binding, and facilitate a re-orientation of CaM N allowing interactions with non-IQ Na V 1.2 residues or auxiliary regulatory proteins interacting in the vicinity of the IQ motif., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Oxidation of methionine disrupts the structure and function of a range of proteins, but little is understood about the chemistry that underlies these perturbations. Using quantum mechanical calculations, we found that oxidation increased the strength of the methionine-aromatic interaction motif, a driving force for protein folding and protein-protein interaction, by 0.5-1.4 kcal/mol. We found that non-hydrogen-bonded interactions between dimethyl sulfoxide (a methionine analog) and aromatic groups were enriched in both the Protein Data Bank and Cambridge Structural Database. Thermal denaturation and NMR spectroscopy experiments on model peptides demonstrated that oxidation of methionine stabilized the interaction by 0.5-0.6 kcal/mol. We confirmed the biological relevance of these findings through a combination of cell biology, electron paramagnetic resonance spectroscopy and molecular dynamics simulations on (i) calmodulin structure and dynamics, and (ii) lymphotoxin-α binding toTNFR1. Thus, the methionine-aromatic motif was a determinant of protein structural and functional sensitivity to oxidative stress.

Synaptotagmin I (Syt I) is a vesicle-localized integral membrane protein that senses the calcium ion (Ca(2+)) influx to trigger fast synchronous release of neurotransmitter. How the cytosolic domains of Syt I allosterically communicate to propagate the Ca(2+) binding signal throughout the protein is not well understood. In particular, it is unclear whether the intrinsically disordered region (IDR) between Syt I's transmembrane helix and first C2 domain (C2A) plays an important role in allosteric modulation of Ca(2+) binding. Moreover, the structural propensity of this IDR with respect to membrane lipid composition is unknown. Using differential scanning and isothermal titration calorimetry, we found that inclusion of the IDR does indeed allosterically modulate Ca(2+) binding within the first C2 domain. Additionally through application of nuclear magnetic resonance, we found that Syt I's IDR interacts with membranes whose lipid composition mimics that of a synaptic vesicle. These findings not only indicate that Syt I's IDR plays a role in regulating Syt I's Ca(2+) sensing but also indicate the IDR is exquisitely sensitive to the underlying membrane lipids. The latter observation suggests the IDR is a key route for communication of lipid organization to the adjacent C2 domains.

Eukaryotic lipids in a bilayer are dominated by weak cooperative interactions. These interactions impart highly dynamic and pliable properties to the membrane. C2 domain-containing proteins in the membrane also interact weakly and cooperatively giving rise to a high degree of conformational plasticity. We propose that this feature of weak energetics and plasticity shared by lipids and C2 domain-containing proteins enhance a cell's ability to transduce information across the membrane. We explored this hypothesis using information theory to assess the information storage capacity of model and mast cell membranes, as well as differential scanning calorimetry, carboxyfluorescein release assays, and tryptophan fluorescence to assess protein and membrane stability. The distribution of lipids in mast cell membranes encoded 5.6-5.8bits of information. More information resided in the acyl chains than the head groups and in the inner leaflet of the plasma membrane than the outer leaflet. When the lipid composition and information content of model membranes were varied, the associated C2 domains underwent large changes in stability and denaturation profile. The C2 domain-containing proteins are therefore acutely sensitive to the composition and information content of their associated lipids. Together, these findings suggest that the maximum flow of signaling information through the membrane and into the cell is optimized by the cooperation of near-random distributions of membrane lipids and proteins. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Objective: Mechanical ventilation with an automated ventilator is recommended during cardiopulmonary resuscitation with a secured airway. We investigated the influence of intermittent positive-pressure ventilation, bilevel ventilation, and the novel ventilator mode chest compression synchronized ventilation, a pressure-controlled ventilation triggered by each chest compression, on gas exchange, hemodynamics, and return of spontaneous circulation in a pig model., Design: Animal study., Setting: University laboratory., Subjects: Twenty-four three-month-old female domestic pigs., Interventions: The study was performed on pigs under general anesthesia with endotracheal intubation. Arterial and central venous catheters were inserted and IV rocuronium (1 mg/kg) was injected. After 3 minutes of cardiac arrest (ventricular fibrillation at t = 0 min), animals were randomized into intermittent positive-pressure ventilation (control group), bilevel, or chest compression synchronized ventilation group. Following 10 minute uninterrupted chest compressions and mechanical ventilation, advanced life support was performed (100% O2, up to six defibrillations, vasopressors)., Measurements and Main Results: Blood gas samples were drawn at 0, 4 and 13 minutes. At 13 minutes, hemodynamics was analyzed beat-to-beat in the end-inspiratory and end-expiratory cycle comparing the IPPV with the bilevel group and the CCSV group. Data were analyzed with the Mann-Whitney U test. Return of spontaneous circulation was achieved in five of eight (intermittent positive-pressure ventilation), six of eight (bilevel), and four of seven (chest compression synchronized ventilation) pigs. The results of arterial blood gas analyses at t = 4 minutes and t = 13 minutes (torr) were as follows: PaO2 intermittent positive-pressure ventilation, 143 (76/256) and 262 (81/340); bilevel, 261 (109/386) (p = 0.195 vs intermittent positive-pressure ventilation) and 236 (86/364) (p = 0.878 vs intermittent positive-pressure ventilation); and chest compression synchronized ventilation, 598 (471/650) (p < 0.001 vs intermittent positive-pressure ventilation) and 634 (115/693) (p = 0.054 vs intermittent positive-pressure ventilation); PaCO2 intermittent positive-pressure ventilation, 40 (38/43) and 45 (36/52); bilevel, 39 (35/41) (p = 0.574 vs intermittent positive-pressure ventilation) and 46 (42/49) (p = 0.798); and chest compression synchronized ventilation, 28 (27/32) (p = 0.001 vs intermittent positive-pressure ventilation) and 26 (18/29) (p = 0.004); mixed venous pH intermittent positive-pressure ventilation, 7.34 (7.31/7.35) and 7.26 (7.25/7.31); bilevel, 7.35 (7.29/7.37) (p = 0.645 vs intermittent positive-pressure ventilation) and 7.27 (7.17/7.31) (p = 0.645 vs intermittent positive-pressure ventilation); and chest compression synchronized ventilation, 7.34 (7.33/7.39) (p = 0.189 vs intermittent positive-pressure ventilation) and 7.35 (7.34/7.36) (p = 0.006 vs intermittent positive-pressure ventilation). Mean end-inspiratory and end-expiratory arterial pressures at t = 13 minutes (mm Hg) were as follows: intermittent positive-pressure ventilation, 28.0 (25.0/29.6) and 27.9 (24.4/30.0); bilevel, 29.1 (25.6/37.1) (p = 0.574 vs intermittent positive-pressure ventilation) and 28.7 (24.2/36.5) (p = 0.721 vs intermittent positive-pressure ventilation); and chest compression synchronized ventilation, 32.7 (30.4/33.4) (p = 0.021 vs intermittent positive-pressure ventilation) and 27.0 (24.5/27.7) (p = 0.779 vs intermittent positive-pressure ventilation)., Conclusions: Both intermittent positive-pressure ventilation and bilevel provided similar oxygenation and ventilation during cardiopulmonary resuscitation. Chest compression synchronized ventilation elicited the highest mean arterial pressure, best oxygenation, and a normal mixed venous pH during cardiopulmonary resuscitation.

Dysferlin plays a critical role in the Ca²⁺-dependent repair of microlesions that occur in the muscle sarcolemma. Of the seven C2 domains in dysferlin, only C2A is reported to bind both Ca²⁺ and phospholipid, thus acting as a key sensor in membrane repair. Dysferlin C2A exists as two isoforms, the "canonical" C2A and C2A variant 1 (C2Av1). Interestingly, these isoforms have markedly different responses to Ca²⁺ and phospholipid. Structural and thermodynamic analyses are consistent with the canonical C2A domain as a Ca²⁺-dependent, phospholipid-binding domain, whereas C2Av1 would likely be Ca²⁺-independent under physiological conditions. Additionally, both isoforms display remarkably low free energies of stability, indicative of a highly flexible structure. The inverted ligand preference and flexibility for both C2A isoforms suggest the capability for both constitutive and Ca²⁺-regulated effector interactions, an activity that would be essential in its role as a mediator of membrane repair., (Copyright © 2014 Elsevier Ltd. All rights reserved.)