N-terminal region is responsible for mHv1 channel activity in MDSCs.

Voltage-gated proton channels (Hv1) are important regulators of the immunosuppressive function of myeloid-derived suppressor cells (MDSCs) in mice and have been proposed as a potential therapeutic target to alleviate dysregulated immunosuppression in tumors. However, till date, there is a lack of evidence regarding the functioning of the Hvcn1 and reports on mHv1 isoform diversity in mice and MDSCs. A computational prediction has suggested that the Hvcn1 gene may express up to six transcript variants, three of which are translated into distinct N-terminal isoforms of mHv1: mHv1.1 (269 aa), mHv1.2 (269 + 42 aa), and mHv1.3 (269 + 4 aa). To validate this prediction, we used RT-PCR on total RNA extracted from MDSCs, and the presence of all six predicted mRNA variances was confirmed. Subsequently, the open-reading frames (ORFs) encoding for mHv1 isoforms were cloned and expressed in Xenopus laevis oocytes for proton current recording using a macro-patch voltage clamp. Our findings reveal that all three isoforms are mammalian mHv1 channels, with distinct differences in their activation properties. Specifically, the longest isoform, mHv1.2, displays a right-shifted conductance-voltage (GV) curve and slower opening kinetics, compared to the mid-length isoform, mHv1.3, and the shortest canonical isoform, mHv1.1. While mHv1.3 exhibits a V0.5 similar to that of mHv1.1, mHv1.3 demonstrates significantly slower activation kinetics than mHv1.1. These results suggest that isoform gating efficiency is inversely related to the length of the N-terminal end. To further explore this, we created the truncated mHv1.2 ΔN20 construct by removing the first 20 amino acids from the N-terminus of mHv1.2. This construct displayed intermediate activation properties, with a V0.5 value lying intermediate of mHv1.1 and mHv1.2, and activation kinetics that were faster than that of mHv1.2 but slower than that of mHv1.1. Overall, these findings indicate that alternative splicing of the N-terminal exon in mRNA transcripts encoding mHv1 isoforms is a regulatory mechanism for mHv1 function within MDSCs. While MDSCs have the capability to translate multiple Hv1 isoforms with varying gating properties, the Hvcn1 gene promotes the dominant expression of mHv1.1, which exhibits the most efficient gating among all mHv1 isoforms.

Functional Site-Directed Fluorometry in Native Cells to Study Skeletal Muscle Excitability.

Functional site-directed fluorometry has been the technique of choice to investigate the structure-function relationship of numerous membrane proteins, including voltage-gated ion channels. This approach has been used primarily in heterologous expression systems to simultaneously measure membrane currents, the electrical manifestation of the channels' activity, and fluorescence measurements, reporting local domain rearrangements. Functional site-directed fluorometry combines electrophysiology, molecular biology, chemistry, and fluorescence into a single wide-ranging technique that permits the study of real-time structural rearrangements and function through fluorescence and electrophysiology, respectively. Typically, this approach requires an engineered voltage-gated membrane channel that contains a cysteine that can be tested by a thiol-reactive fluorescent dye. Until recently, the thiol-reactive chemistry used for the site-directed fluorescent labeling of proteins was carried out exclusively in Xenopus oocytes and cell lines, restricting the scope of the approach to primary non-excitable cells. This report describes the applicability of functional site-directed fluorometry in adult skeletal muscle cells to study the early steps of excitation-contraction coupling, the process by which muscle fiber electrical depolarization is linked to the activation of muscle contraction. The present protocol describes the methodologies to design and transfect cysteine-engineered voltage-gated Ca2+ channels (CaV1.1) into muscle fibers of the flexor digitorum brevis of adult mice using in vivo electroporation and the subsequent steps required for functional site-directed fluorometry measurements. This approach can be adapted to study other ion channels and proteins. The use of functional site-directed fluorometry of mammalian muscle is particularly relevant to studying basic mechanisms of excitability.

Voltage sensor movements of CaV1.1 during an action potential in skeletal muscle fibers.

The skeletal muscle L-type Ca2+ channel (CaV1.1) works primarily as a voltage sensor for skeletal muscle action potential (AP)-evoked Ca2+ release. CaV1.1 contains four distinct voltage-sensing domains (VSDs), yet the contribution of each VSD to AP-evoked Ca2+ release remains unknown. To investigate the role of VSDs in excitation-contraction coupling (ECC), we encoded cysteine substitutions on each S4 voltage-sensing segment of CaV1.1, expressed each construct via in vivo gene transfer electroporation, and used in cellulo AP fluorometry to track the movement of each CaV1.1 VSD in skeletal muscle fibers. We first provide electrical measurements of CaV1.1 voltage sensor charge movement in response to an AP waveform. Then we characterize the fluorescently labeled channels' VSD fluorescence signal responses to an AP and compare them with the waveforms of the electrically measured charge movement, the optically measured free myoplasmic Ca2+, and the calculated rate of Ca2+ release from the sarcoplasmic reticulum for an AP, the physiological signal for skeletal muscle fiber activation. A considerable fraction of the fluorescence signal for each VSD occurred after the time of peak Ca2+ release, and even more occurred after the earlier peak of electrically measured charge movement during an AP, and thus could not directly reflect activation of Ca2+ release or charge movement, respectively. However, a sizable fraction of the fluorometric signals for VSDs I, II, and IV, but not VSDIII, overlap the rising phase of charge moved, and even more for Ca2+ release, and thus could be involved in voltage sensor rearrangements or Ca2+ release activation.

Mechanoactivation of NOX2-generated ROS elicits persistent TRPM8 Ca2+ signals that are inhibited by oncogenic KRas.

Changes in the mechanical microenvironment and mechanical signals are observed during tumor progression, malignant transformation, and metastasis. In this context, understanding the molecular details of mechanotransduction signaling may provide unique therapeutic targets. Here, we report that normal breast epithelial cells are mechanically sensitive, responding to transient mechanical stimuli through a two-part calcium signaling mechanism. We observed an immediate, robust rise in intracellular calcium (within seconds) followed by a persistent extracellular calcium influx (up to 30 min). This persistent calcium was sustained via microtubule-dependent mechanoactivation of NADPH oxidase 2 (NOX2)-generated reactive oxygen species (ROS), which acted on transient receptor potential cation channel subfamily M member 8 (TRPM8) channels to prolong calcium signaling. In contrast, the introduction of a constitutively active oncogenic KRas mutation inhibited the magnitude of initial calcium signaling and severely blunted persistent calcium influx. The identification that oncogenic KRas suppresses mechanically-induced calcium at the level of ROS provides a mechanism for how KRas could alter cell responses to tumor microenvironment mechanics and may reveal chemotherapeutic targets for cancer. Moreover, we find that expression changes in both NOX2 and TRPM8 mRNA predict poor clinical outcome in estrogen receptor (ER)-negative breast cancer patients, a population with limited available treatment options. The clinical and mechanistic data demonstrating disruption of this mechanically-activated calcium pathway in breast cancer patients and by KRas activation reveal signaling alterations that could influence cancer cell responses to the tumor mechanical microenvironment and impact patient survival.

LRP1 (Low-Density Lipoprotein Receptor-Related Protein 1) Regulates Smooth Muscle Contractility by Modulating Ca2+ Signaling and Expression of Cytoskeleton-Related Proteins.

Objective- Mutations affecting contractile-related proteins in the ECM (extracellular matrix), microfibrils, or vascular smooth muscle cells can predispose the aorta to aneurysms. We reported previously that the LRP1 (low-density lipoprotein receptor-related protein 1) maintains vessel wall integrity, and smLRP1-/- mice exhibited aortic dilatation. The current study focused on defining the mechanisms by which LRP1 regulates vessel wall function and integrity. Approach and Results- Isometric contraction assays demonstrated that vasoreactivity of LRP1-deficient aortic rings was significantly attenuated when stimulated with vasoconstrictors, including phenylephrine, thromboxane receptor agonist U-46619, increased potassium, and L-type Ca2+ channel ligand FPL-64176. Quantitative proteomics revealed proteins involved in actin polymerization and contraction were significantly downregulated in aortas of smLRP1-/- mice. However, studies with calyculin A indicated that although aortic muscle from smLRP1-/- mice can contract in response to calyculin A, a role for LRP1 in regulating the contractile machinery is not revealed. Furthermore, intracellular calcium imaging experiments identified defects in calcium release in response to a RyR (ryanodine receptor) agonist in smLRP1-/- aortic rings and cultured vascular smooth muscle cells. Conclusions- These results identify a critical role for LRP1 in modulating vascular smooth muscle cell contraction by regulating calcium signaling events that potentially protect against aneurysm development.

Real-time scratch assay reveals mechanisms of early calcium signaling in breast cancer cells in response to wounding.

Aggressive cellular phenotypes such as uncontrolled proliferation and increased migration capacity engender cellular transformation, malignancy and metastasis. While genetic mutations are undisputed drivers of cancer initiation and progression, it is increasingly accepted that external factors are also playing a major role. Two recently studied modulators of breast cancer are changes in the cellular mechanical microenvironment and alterations in calcium homeostasis. While many studies investigate these factors separately in breast cancer cells, very few do so in combination. This current work sets a foundation to explore mechano-calcium relationships driving malignant progression in breast cancer. Utilizing real-time imaging of an in vitro scratch assay, we were able to resolve mechanically-sensitive calcium signaling in human breast cancer cells. We observed rapid initiation of intracellular calcium elevations within seconds in cells at the immediate wound edge, followed by a time-dependent increase in calcium in cells at distances up to 500µm from the scratch wound. Calcium signaling to neighboring cells away from the wound edge returned to baseline within seconds. Calcium elevations at the wound edge however, persisted for up to 50 minutes. Rigorous quantification showed that extracellular calcium was necessary for persistent calcium elevation at the wound edge, but intercellular signal propagation was dependent on internal calcium stores. In addition, intercellular signaling required extracellular ATP and activation of P2Y2 receptors. Through comparison of scratch-induced signaling from multiple cell lines, we report drastic reductions in response from aggressively tumorigenic and metastatic cells. The real-time scratch assay established here provides quantitative data on the molecular mechanisms that support rapid scratch-induced calcium signaling in breast cancer cells. These mechanisms now provide a clear framework for investigating which short-term calcium signals promote long-term changes in cancer cell biology.

Impaired calcium signaling in muscle fibers from intercostal and foot skeletal muscle in a cigarette smoke-induced mouse model of COPD.

INTRODUCTION: Respiratory and locomotor skeletal muscle dysfunction are common findings in chronic obstructive pulmonary disease (COPD); however, the mechanisms that cause muscle impairment in COPD are unclear. Because Ca2+ signaling in excitation-contraction (E-C) coupling is important for muscle activity, we hypothesized that Ca2+ dysregulation could contribute to muscle dysfunction in COPD. METHODS: Intercostal and flexor digitorum brevis muscles from control and cigarette smoke-exposed mice were investigated. We used single cell Ca2+ imaging and Western blot assays to assess Ca2+ signals and E-C coupling proteins. RESULTS: We found impaired Ca2+ signals in muscle fibers from both muscle types, without significant changes in releasable Ca2+ or in the expression levels of E-C coupling proteins. CONCLUSIONS: Ca2+ dysregulation may contribute or accompany respiratory and locomotor muscle dysfunction in COPD. These findings are of significance to the understanding of the pathophysiological course of COPD in respiratory and locomotor muscles. Muscle Nerve 56: 282-291, 2017.

Elevated nuclear Foxo1 suppresses excitability of skeletal muscle fibers.

Forkhead box O 1 (Foxo1) controls the expression of proteins that carry out processes leading to skeletal muscle atrophy, making Foxo1 of therapeutic interest in conditions of muscle wasting. The transcription of Foxo1-regulated proteins is dependent on the translocation of Foxo1 to the nucleus, which can be repressed by insulin-like growth factor-1 (IGF-1) treatment. The role of Foxo1 in muscle atrophy has been explored at length, but whether Foxo1 nuclear activity affects skeletal muscle excitation-contraction (EC) coupling has not yet been examined. Here, we use cultured adult mouse skeletal muscle fibers to investigate the effects of Foxo1 overexpression on EC coupling. Fibers expressing Foxo1-green fluorescent protein (GFP) exhibit an inability to contract, impaired propagation of action potentials, and ablation of calcium transients in response to electrical stimulation compared with fibers expressing GFP alone. Evaluation of the transverse (T)-tubule system morphology, the membranous system involved in the radial propagation of the action potential, revealed an intact T-tubule network in fibers overexpressing Foxo1-GFP. Interestingly, long-term IGF-1 treatment of Foxo1-GFP fibers, which maintains Foxo1-GFP outside the nucleus, prevented the loss of normal calcium transients, indicating that Foxo1 translocation and the atrogenes it regulates affect the expression of proteins involved in the generation and/or propagation of action potentials. A reduction in the sodium channel Nav1.4 expression in fibers overexpressing Foxo1-GFP was also observed in the absence of IGF-1. We conclude that increased nuclear activity of Foxo1 prevents the normal muscle responses to electrical stimulation and that this indicates a novel capability of Foxo1 to disable the functional activity of skeletal muscle.

NOX2-dependent ROS is required for HDAC5 nuclear efflux and contributes to HDAC4 nuclear efflux during intense repetitive activity of fast skeletal muscle fibers.

Reactive oxygen species (ROS) have been linked to oxidation and nuclear efflux of class IIa histone deacetylase 4 (HDAC4) in cardiac muscle. Here we use HDAC-GFP fusion proteins expressed in isolated adult mouse flexor digitorum brevis muscle fibers to study ROS mediation of HDAC localization in skeletal muscle. H(2)O(2) causes nuclear efflux of HDAC4-GFP or HDAC5-GFP, which is blocked by the ROS scavenger N-acetyl-l-cysteine (NAC). Repetitive stimulation with 100-ms trains at 50 Hz, 2/s ("50-Hz trains") increased ROS production and caused HDAC4-GFP or HDAC5-GFP nuclear efflux. During 50-Hz trains, HDAC5-GFP nuclear efflux was completely blocked by NAC, but HDAC4-GFP nuclear efflux was only partially blocked by NAC and partially blocked by the calcium-dependent protein kinase (CaMK) inhibitor KN-62. Thus, during intense activity both ROS and CaMK play roles in nuclear efflux of HDAC4, but only ROS mediates HDAC5 nuclear efflux. The 10-Hz continuous stimulation did not increase the rate of ROS production and did not cause HDAC5-GFP nuclear efflux but promoted HDAC4-GFP nuclear efflux that was sensitive to KN-62 but not NAC and thus mediated by CaMK but not by ROS. Fibers from NOX2 knockout mice lacked ROS production and ROS-dependent nuclear efflux of HDAC5-GFP or HDAC4-GFP during 50-Hz trains but had unmodified Ca(2+) transients. Our results demonstrate that ROS generated by NOX2 could play important roles in muscle remodeling due to intense muscle activity and that the nuclear effluxes of HDAC4 and HDAC5 are differentially regulated by Ca(2+) and ROS during muscle activity.

Effects of conformational peptide probe DP4 on bidirectional signaling between DHPR and RyR1 calcium channels in voltage-clamped skeletal muscle fibers.

In skeletal muscle, excitation-contraction coupling involves the activation of dihydropyridine receptors (DHPR) and type-1 ryanodine receptors (RyR1) to produce depolarization-dependent sarcoplasmic reticulum Ca²âº release via orthograde signaling. Another form of DHPR-RyR1 communication is retrograde signaling, in which RyRs modulate the gating of DHPR. DP4 (domain peptide 4), is a peptide corresponding to residues Leu²44²-Pro²477 of the central domain of the RyR1 that produces RyR1 channel destabilization. Here we explore the effects of DP4 on orthograde excitation-contraction coupling and retrograde RyR1-DHPR signaling in isolated murine muscle fibers. Intracellular dialysis of DP4 increased the peak amplitude of Ca²âº release during step depolarizations by 64% without affecting its voltage-dependence or kinetics, and also caused a similar increase in Ca²âº release during an action potential waveform. DP4 did not modify either the amplitude or the voltage-dependence of the intramembrane charge movement. However, DP4 augmented DHPR Ca²âº current density without affecting its voltage-dependence. Our results demonstrate that the conformational changes induced by DP4 regulate both orthograde E-C coupling and retrograde RyR1-DHPR signaling.

S100A1 binds to the calmodulin-binding site of ryanodine receptor and modulates skeletal muscle excitation-contraction coupling.

S100A1, a 21-kDa dimeric Ca2+-binding protein, is an enhancer of cardiac Ca2+ release and contractility and a potential therapeutic agent for the treatment of cardiomyopathy. The role of S100A1 in skeletal muscle has been less well defined. Additionally, the precise molecular mechanism underlying S100A1 modulation of sarcoplasmic reticulum Ca2+ release in striated muscle has not been fully elucidated. Here, utilizing a genetic approach to knock out S100A1, we demonstrate a direct physiological role of S100A1 in excitation-contraction coupling in skeletal muscle. We show that the absence of S100A1 leads to decreased global myoplasmic Ca2+ transients following electrical excitation. Using high speed confocal microscopy, we demonstrate with high temporal resolution depressed activation of sarcoplasmic reticulum Ca2+ release in S100A1-/- muscle fibers. Through competition assays with sarcoplasmic reticulum vesicles and through tryptophan fluorescence experiments, we also identify a novel S100A1-binding site on the cytoplasmic face of the intact ryanodine receptor that is conserved throughout striated muscle and corresponds to a previously identified calmodulin-binding site. Using a 12-mer peptide of this putative binding domain, we demonstrate low micromolar binding affinity to S100A1. NMR spectroscopy reveals this peptide binds within the Ca2+-dependent hydrophobic pocket of S100A1. Taken together, these data suggest that S100A1 plays a significant role in skeletal muscle excitation-contraction coupling, primarily through specific interactions with a conserved binding domain of the ryanodine receptor. This warrants further investigation into the use of S100A1 as a therapeutic target for the treatment of both cardiac and skeletal myopathies.

Ca2+ signal summation and NFATc1 nuclear translocation in sympathetic ganglion neurons during repetitive action potentials.

NFATc-mediated gene expression constitutes a critical step during neuronal development and synaptic plasticity. Although considerable information is available regarding the activation and functionality of specific NFATc isoforms, in neurons little is known about how sensitive NFAT nuclear translocation is to specific patterns of electrical activity. Here we used high-speed fluo-4 confocal imaging to monitor action potential (AP)-induced cytosolic Ca2+ transients in rat sympathetic neurons. We have recorded phasic and repetitive AP patterns, and corresponding Ca2+ transients initiated by either long (100-800 ms) current-clamp pulses, or single brief (2 ms) electrical field stimulation. We address the functional consequences of these AP and Ca2+ transient patterns, by using an adenoviral construct to express NFATc1-CFP and evaluate NFATc1-CFP nuclear translocation in response to specific patterns of electrical activity. Ten Hertz trains stimulation induced nuclear translocation of NFATc1, whereas 1 Hz trains did not. However, 1 Hz train stimulation did result in NFATc1 translocation in the presence of 2 mM Ba2+, which inhibits M-currents and promotes repetitive firing and the accompanying small (approximately 0.6 DeltaF/F0) repetitive and summating Ca2+ transients. Our results demonstrate that M-current inhibition-mediated spike frequency facilitation enhances cytosolic Ca2+ signals and NFATc1 nuclear translocation during trains of low frequency electrical stimulation.