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Ca II emission-line surface fluxes are derived for 14 stars from 17 A/mm photographic spectra. Most of the stars observed are active chromosphere binaries; a few are known X-ray sources or have been observed by the IUE. The status of optical information on each of the objects is reviewed, and new information on v sin i and duplicity is presented.
Spikes in free Ca(2+) initiate contractions in skeletal muscle cells, but whether and how they might signal to transcription factors in skeletal muscles of living animals is unknown. Since previous studies in non-muscle cells have shown that serum response factor (SRF) protein, a transcription factor, is phosphorylated rapidly by Ca(2+)/calmodulin (CaM)-dependent protein kinase after rises in intracellular Ca(2+), we measured enzymatic activity that phosphorylates SRF (designated SRF kinase activity). Homogenates from 7-day-hypertrophied anterior latissimus dorsi muscles of roosters had more Ca(2+)-independent SRF kinase activity than their respective control muscles. However, no differences were noted in Ca(2+)/CaM-dependent SRF kinase activity between control and trained muscles. To determine whether the Ca(2+)-independent and Ca(2+)/CaM-dependent forms of Ca(2+)/CaM-dependent protein kinase II (CaMKII) might contribute to some of the SRF kinase activity, autocamtide-3, a synthetic substrate that is specific for CaMKII, was employed. While the Ca(2+)-independent form of CaMKII was increased, like the Ca(2+)-independent form of SRF kinase, no alteration in CaMKII occurred at 7 days of stretch overload. These observations suggest that some of SRF phosphorylation by skeletal muscle extracts could be due to CaMKII. To determine whether this adaptation was specific to the exercise type (i.e., hypertrophy), similar measurements were made in the white vastus lateralis muscle of rats that had completed 2 wk of voluntary running. Although Ca(2+)-independent SRF kinase was increased, no alteration occurred in Ca(2+)/CaM-dependent SRF kinase activity. Thus any role of Ca(2+)-independent SRF kinase signaling has downstream modulators specific to the exercise phenotype.
Large-conductance Ca 2+ and voltage-activated K + (BK) channels control membrane excitability in many cell types. BK channels are tetrameric. Each subunit is composed of a voltage sensor domain (VSD), a central pore-gate domain, and a large cytoplasmic domain (CTD) that contains the Ca 2+ sensors. While it is known that BK channels are activated by voltage and Ca 2+ , and that voltage and Ca 2+ activations interact, less is known about the mechanisms involved. In this work we explore these mechanisms by examining the gating contribution of an interface formed between the VSDs and the αB helices located at the top of the CTDs. Proline mutations in the αB helix greatly decreased voltage activation while having negligible effects on gating currents. Analysis with the Horrigan, Cui, and Aldrich model indicated a decreased coupling between voltage sensors and pore gate. Proline mutations decreased Ca 2+ activation for both Ca 2+ bowl and RCK1 Ca 2+ sites, suggesting that both high-affinity Ca 2+ sites transduce their effect, at least in part, through the αB helix. Mg 2+ activation also decreased. The crystal structure of the CTD with proline mutation L390P showed a flattening of the first helical turn in the αB helix compared to wild type, without other notable differences in the CTD, indicating that structural changes from the mutation were confined to the αB helix. These findings indicate that an intact αB helix/VSD interface is required for effective coupling of Ca 2+ binding and voltage depolarization to pore opening and that shared Ca 2+ and voltage transduction pathways involving the αB helix may be involved.
Ca(2+)-dependent protein kinases (CDPK) have a calmodulin-like domain (CaM-LD) tethered to the C-terminal end of the kinase. Activation is proposed to involve intramolecular binding of the CaM-LD to a junction sequence that connects the CaM-LD to the kinase domain. Consistent with this model, a truncated CDPK (DeltaNC) in which the CaM-LD has been deleted can be activated in a bimolecular interaction with an isolated CaM-LD or calmodulin, similar to the activation of a calmodulin-dependent protein kinase (CaMK) by calmodulin. Here we provide genetic evidence that this bimolecular activation requires a nine-residue binding segment from F436 to I444 (numbers correspond to CPK-1 accession number L14771). Two mutations at either end of this core segment (F436/A and VI444/AA) severely disrupted bimolecular activation, whereas flanking mutations had only minor effects. Intramolecular activation of a full-length kinase was also disrupted by a VI444/AA mutation, but surprisingly not by a F436/A mutation (at the N-terminal end of the binding site). Interestingly, intramolecular but not bimolecular activation was disrupted by insertion mutations placed immediately downstream of I444. To show that mutant enzymes were not misfolded, latent kinase activity was stimulated through binding of an antijunction antibody. Results here support a model of intramolecular activation in which the tether (A445 to G455) that connects the CaM-LD to the kinase provides an important structural constraint and is not just a simple flexible connection.
Ca(2+)-dependent protein kinases (CDPKs) are regulated by a C-terminal calmodulin-like domain (CaM-LD). The CaM-LD is connected to the kinase by a short junction sequence which contains a pseudosubstrate autoinhibitor. To understand how the CaM-LD regulates a CDPK, a recombinant CDPK (isoform CPK-1 from Arabidopsis, accession no. L14771) was made as a fusion protein in Escherichia coli. We show here that a truncated CDPK lacking a CaM-LD (e.g. mutant delta NC-26H) can be activated by exogenous calmodulin or an isolated CaM-LD (Kact approximately 2 microM). We propose that Ca2+ activation of a CDPK normally occurs through intramolecular binding of the CaM-LD to the junction. When the junction and CaM-LD are made as two separate polypeptides, the CaM-LD can bind the junction in a Ca(2+)-dependent fashion with a dissociation constant (KD) of 6 x 10(-6) M, as determined by kinetic binding analyses. When the junction and CaM-LD are tethered in a single polypeptide (e.g. in protein JC-1), their ability to engage in bimolecular binding is suppressed (e.g. the tethered CaM-LD cannot bind a separate junction). A mutation which disrupts the putative CaM-LD binding sequence (e.g. substitution LRV-1444 to DLPG) appears to block intramolecular binding, as indicated by the restored ability of a tethered CaM-LD to engage in bimolecular binding. This mutation, in the context of a full-length enzyme (mutant KJM46H), appears to block Ca2+ activation. Thus, a disruption of intramolecular binding correlates with a disruption of the Ca2+ activation mechanism. CDPKs provide the first example of a member of the calmodulin superfamily where a target binding sequence is located within the same polypeptide.
Missense variant Ile79Asn in human cardiac troponin T (cTnT-I79N) has been associated with hypertrophic cardiomyopathy and sudden cardiac arrest in juveniles. cTnT-I79N is located in the cTnT N-terminal (TnT1) loop region and is known for its pathological and prognostic relevance. A recent structural study revealed that I79 is part of a hydrophobic interface between the TnT1 loop and actin, which stabilizes the relaxed (OFF) state of the cardiac thin filament. Given the importance of understanding the role of TnT1 loop region in Ca 2+ regulation of the cardiac thin filament along with the underlying mechanisms of cTnT-I79N-linked pathogenesis, we investigated the effects of cTnT-I79N on cardiac myofilament function. Transgenic I79N (Tg-I79N) muscle bundles displayed increased myofilament Ca 2+ sensitivity, smaller myofilament lattice spacing, and slower crossbridge kinetics. These findings can be attributed to destabilization of the cardiac thin filament’s relaxed state resulting in an increased number of crossbridges during Ca 2+ activation. Additionally, in the low Ca 2+ -relaxed state (pCa8), we showed that more myosin heads are in the disordered-relaxed state (DRX) that are more likely to interact with actin in cTnT-I79N muscle bundles. Dysregulation of the myosin super-relaxed state (SRX) and the SRX/DRX equilibrium in cTnT-I79N muscle bundles likely result in increased mobility of myosin heads at pCa8, enhanced actomyosin interactions as evidenced by increased active force at low Ca 2+ , and increased sinusoidal stiffness. These findings point to a mechanism whereby cTnT-I79N weakens the interaction of the TnT1 loop with the actin filament, which in turn destabilizes the relaxed state of the cardiac thin filament.
The ability of interferon-gamma (IFN-gamma) to alter cytoplasmic Ca(2+) content in the monocytelike cell line U937 was investigated, using a slow Ca-channel blocker, diltiazem. In addition, the Ca-ATPase and the Ca-uptake activities were measured in isolated U937 membranes, together with the effect of inositol trisphosphate (IP3) upon the Ca(2+) release from Ca-loaded membranes. The addition of 50 U/ml INF-gamma to U937 cultures was found to increase internal Ca(2+) by about 100 percent within 3 min. The increase was significantly reduced by incubation in Ca-free buffer or by the addition of diltiazem. A crude membrane preparation from U937 cells was found to contain significant amounts of Ca-ATPase activity and to sequester Ca(2+) to a level of 8 nmol/mg in 30 sec; the addition of IP3 induced release of a portion of the sequestered Ca(2+) which was then resequestered. The results suggest that IFN-gamma causes an increase of cytoplasmic Ca(2+), in part, by the IP3-induced release from the internal storage sites and, in part, from the entry of extracellular Ca through slow channels.
Metacaspases are highly conserved in plants and play essential roles in mediating programmed cell death, biotic and abiotic stress responses, and damage-induced innate immunity. Ca 2+ signaling induced by plant damage leads to activation of metacaspase from Arabidopsis thaliana (AtMC4), which subsequently processes a plant elicitor peptide to trigger downstream immuno-response. To understand the structural basis of AtMC4 activation by Ca 2+ , we previously determined its crystal structure and performed in-crystal Ca 2+ treatment to probe activation-associated conformational changes. To enable structure determination and in-crystal Ca 2+ activation analysis, we used microcrystals and related methods which were essential for our successful approach. Here, in this paper, we describe in detail the methods that we used for determination of AtMC4 structure using single-wavelength isomorphous replacement with anomalous signals assembled from 22 microcrystals. We also describe the method for in-crystal Ca 2+ soaking, microcrystal data collection, data assembly and analysis to obtain the activated structure of AtMC4 from 91 micro-sized crystals. The described methods may be useful to study other plant metacaspases and more broadly other plant enzymes for their structure determination and in-crystal functional characterization.
The 1,4-dihydropyridines, drugs with well-established bioavailability and toxicity profiles, have proven efficacy in treating human hypertension, peripheral vascular disorders, and coronary artery disease. Every 1,4-dihydropyridine in clinical use blocks L-type voltage-gated calcium channels. We now report our development, using selective optimization of a side activity (SOSA), of a class of 1,4-dihydropyridines that selectively and potently inhibit the intermediate-conductance calcium-activated K + channel K Ca 3.1, a validated therapeutic target for diseases affecting many organ systems. One of these 1,4-dihydropyridines, DHP-103, blocked K Ca 3.1 with an IC 50 of 6 nM and exhibited exquisite selectivity over calcium channels and a panel of >100 additional molecular targets. Using high-resolution structure determination by cryogenic electron microscopy together with mutagenesis and electrophysiology, we delineated the drug binding pocket for DHP-103 within the water-filled central cavity of the K Ca 3.1 channel pore, where bound drug directly impedes ion permeation. DHP-103 inhibited gain-of-function mutant K Ca 3.1 channels that cause hereditary xerocytosis, suggesting its potential use as a therapeutic for this hemolytic anemia. In a rat model of acute ischemic stroke, the second leading cause of death worldwide, DHP-103 administered 12 h postischemic insult in proof-of-concept studies reduced infarct volume, improved balance beam performance (measure of proprioception) and decreased numbers of activated microglia in infarcted areas. K Ca 3.1-selective 1,4-dihydropyridines hold promise for the many diseases for which K Ca 3.1 has been experimentally confirmed as a therapeutic target.
A chimeric Ca(2+)/calmodulin-dependent protein kinase (CCaMK) gene characterized by a catalytic domain, a calmodulin-binding domain, and a neural visinin-like Ca(2+)-binding domain was recently cloned from plants. The Escherichia coli-expressed CCaMK phosphorylates various protein and peptide substrates in a Ca(2+)/calmodulin-dependent manner. The calmodulin-binding region of CCAMK has similarity to the calmodulin-binding region of the alpha-subunit of multifunctional Ca(2+)/calmodulin-dependent protein kinase (CaMKII). CCaMK exhibits basal autophosphorylation at the threonine residue(s) (0.098 mol of P-32/mol) that is stimulated 3.4-fold by Ca(2+) (0.339 mol of P-32/mol), while calmodulin inhibits Ca(2+)-stimulated autophosphorylation to the basal level. A deletion mutant lacking the visinin-like domain did not show Ca(2+)-simulated autophosphorylation activity but retained Ca(2+)/calmodulin-dependent protein kinase activity at a reduced level. Ca(2+)-dependent mobility shift assays using E.coli-expressed protein from residues 358-520 revealed that Ca(2+) binds to the visinin-like domain. Studies with site-directed mutants of the visinin-like domain indicated that EF-hands II and III are crucial for Ca(2+)-induced conformational changes in the visinin-like domain. Autophosphorylation of CCaMK increases Ca(2+)/calmodulin-dependent protein kinase activity by about 5-fold, whereas it did not affect its C(2+)-independent activity. This report provides evidence for the existence of a protein kinase in plants that is modulated by Ca(2+) and Ca(2+)/calmodulin. The presence of a visinin-like Ca(2+)-binding domain in CCaMK adds an additional Ca(2+)-sensing mechanism not previously known to exist in the Ca(2+)/calmodulin-mediated signaling cascade in plants.
Myosin cross-bridges dissociate from actin following Mg 2+ -adenosine triphosphate (MgATP) binding. Myosin hydrolyses MgATP into inorganic phosphate (P i ) and Mg 2+ -adenosine diphosphate (ADP), and release of these hydrolysis products drives chemo-mechanical energy transitions within the cross-bridge cycle to power muscle contraction. Some forms of heart disease are associated with metabolic or enzymatic dysregulation of the MgATP-MgADP nucleotide pool, resulting in elevated cytosolic [MgADP] and impaired muscle relaxation. We investigated the mechanical and structural effects of increasing [MgADP] in permeabilized myocardial strips from porcine left ventricle samples. Sarcomere length was set to 2.0 µm at 28 °C, and all solutions contained 3% dextran T-500 to compress myofilament lattice spacing to near-physiological values. Under relaxing low [Ca 2+ ] conditions (pCa 8.0, where pCa = -log 10 [Ca 2+ ]), tension increased as [MgADP] increased from 0-5 mM. Complementary small-angle X-ray diffraction measurements show that the equatorial intensity ratio, I 1,1 /I 1,0 , also increased as [MgADP] increased from 0 to 5 mM, indicating myosin head movement away from the thick-filament backbone towards the thin-filament. Ca 2+ -activated force-pCa measurements show that Ca 2+ -sensitivity of contraction increased with 5 mM MgADP, compared to 0 mM MgADP. These data show that MgADP augments tension at low [Ca 2+ ] and Ca 2+ -sensitivity of contraction, suggesting that MgADP destabilizes the quasi-helically ordered myosin OFF state, thereby shifting the cross-bridge population towards the disordered myosin ON state. Together, these results indicate that MgADP enhances the probability of cross-bridge binding to actin due to enhancement of both thick and thin filament-based activation mechanisms.
The bullfrog saccule, a sensor of gravity and substrate-borne vibration, is a model system for hair cell transduction. Saccular hair cells also increase in number throughout adult life and rapidly recover after hair cell damage, making this organ an ideal system for studying hair cell development, repair, and regeneration. We have used of hair cell and supporting cell immunocytochemical markers to identify damaged hair cells and hair cell precursors in organotypic cultures of the bullfrog saccule. We then used an innovative combination of confocal, electron, and time-lapse microscopy to study the fate of damaged hair cells and the origin of new hair cells after gentamicin ototoxicity in normal and mitotically blocked saccular cultures. These studies have shown that gentamicin ototoxicity produces both lethal and sublethal hair cell damage. They have also shown that hair cell recovery in this organ takes place by both the repair of sublethally damaged hair cells and by the replacement of lost hair cells by mitotic regeneration. In parallel studies, we have used biophysical and molecular biological techniques to study the differentiation and innervation of developing, repairing, and regenerating hair cells. More specifically, we have used RT-PCR to obtain the bullfrog homologues of L-type voltage- gated calcium (L-VGCC) and large-conductance Ca(2+)-activated potassium (BK) channel genes. We have then obtained probes for these genes and, using in situ hybridization, begun to examine their expression in the bullfrog saccule and amphibian papilla. We have also used fluorescent-labeled channel toxins and channel toxin derivatives to determine the time of appearance of L-type voltage-gated calcium (L-VGCC) and Ca(2+)-activated potassium (BK) channels and to study dynamic changes in the number, distribution, and co-localization of these proteins in developing, repairing, and regenerating hair cells. Using time-lapse microscopy, we are also studying the dynamic relationship between ion channel clustering and synaptic formation in hair cells and afferent neurons. In future studies, we will determine when hair cell precursors acquire electrical tuning, and, using whole-cell patch-clamp techniques, identify and characterize their L-VGCC and BK currents. We will also use biophysical techniques to determine the number of L-VGCC and BK channels and the size and gating kinetics of their underlying L-VGCC and BK conductances, correlating these variables with the amplitude and frequency of membrane oscillations produced by intracellular current steps. We expect these studies to determine how hair cells regulate ion channel expression to achieve specific physiological responses.
In rat dorsal root ganglia, histochemical staining of carbonic anhydrase (CA) and cholinesterase (CE) yields a reciprocal pattern of activity: Sensory processes are CA positive and CE negative, whereas motor processes are CA negative and CE positive. In rat infraorbital nerve (a sensory peripheral nerve), we saw extensive CA staining of nearly 100% of the myelinated axons. Although CE reactivity in myelinated axons was extremely rare, we did observe CE staining of unmyelinated autonomic fibers. Four weeks after transection of infraorbital nerves, CA-stained longitudinal sections of the proximal stump demonstrated 3 distinct morphological zones. A fraction of the viable axons retained CA activity to within 2 mm of the distal extent of the stump, and the stain is capable of resolving growth sprouts being regenerated from these fibers. Staining of unmyelinated autonomic fibers in serial sections shows that CE activity was not retained as far distally as is the CA sensory staining.
The utilization of oxide frameworks as intercalation cathodes for non-aqueous Ca-ion batteries potentially unlocks a new energy storage system that delivers high energy density. However, the slow kinetics of Ca 2+ in oxide electrodes strongly handicaps their activity and reversibility at room temperature. Here, nanocrystals of layered MnO x containing a high concentration of atomic defects and lattice water are shown to have remarkable electrochemical activity towards Ca 2+ , amounting to a capacity of ~130 mAh/g at room temperature. Multimodal characterization revealed the notable degree of intercalation by probing the structural, compositional and redox changes undertaken by the defective MnO x nanocrystals. The results suggest that the existence of atomic defects and lattice water played a role in improving Ca 2+ diffusivity in the oxide. These outcomes reaffirm the prospects for functional Ca-ion batteries using oxide cathodes under moderate conditions.
The reduction of protein content in skeletal muscle undergoing disuse-induced atrophy is correlated with accelerated rates of protein degradation and reduced rates of protein synthesis (Goldspink, 1977). It is not known in what manner myofibers are partially disassembled during disuse atrophy to fibers of smaller diameter; nor is it known which proteases are responsible for this morphological change in contractile protein mass. Dayton and colleagues (1975) have suggested that the Ca(2+)-activated protease (CaP) may initiate myofibril degradation. The discovery of a form of CaP that is activatable by nano-molar concentrations of Ca(2+) indicates that CaP activity may be regulated by physiological concentrations of Ca(2+) (Mellgren, 1980). The enhancement of proteolysis by the Ca(2+) ionophore A23187, reported by Etlinger (1979), is consistent with a significant role for CaP in protein degradation. It was of interest, therefore, to measure the levels of CaP activity and the CaP inhibitor in extracts obtained from skeletal muscles of rat and chicken limbs undergoing disuse atrophy or stretch hypertrophy, respectively.
New observations of the Ca II H and K and H-epsilon region and/or the Balmer H-alpha line are presented for 100 mostly very active stars but also for weak or inactive stars with suspected activity. Correlations between chromospheric activity at Ca II H and K and H-alpha and effective surface temperature and rotation are identified, and several new stars with chromospheric Ca II H and K emission are discovered. No single activity-rotation relation can be derived for all luminosity classes, and there is clear evidence that evolved stars are generally more active than main-sequence stars of the same rotation period. Binary within the evolved stars appears to play no role, while main-sequence binary stars show generally higher levels of activity than their single counterparts. Chromospheric emission in the Ca II H and K lines depends on surface temperature in that flux declines with cooler temperature.
Spectroscopic observations of the Ca II infrared triplet (8498, 8542, 8662 A) have been obtained for 45 stars which are known or suspected to be chromospherically active. The sample includes both single and binary stars of spectral types from F2 to M5 spanning luminosity classes III, IV, and V. Several different types of activity diagnostics were measured, and their relative merits are discussed. Dependence of chromospheric emission upon rotation period, luminosity, temperature, and duplicity are analyzed. Synchronous binaries show a slight trend of increased emission with decreasing period while the asynchronous binaries show abnormally high activity levels for their rotation periods. Several stars exhibit rotationally modulated emission which is anticorrelated with the stellar brightness. Finally, estimates of chromospheric energy losses are presented with the result that the total loss in the infrared triplet is about twice that of the H and K lines.
TMEM16F is a Ca 2+ -activated phospholipid scramblase in the TMEM16 family of membrane proteins. Unlike other TMEM16s exhibiting a membrane-exposed hydrophilic groove that serves as a translocation pathway for lipids, the experimentally determined structures of TMEM16F shows the groove in a closed conformation even under conditions of maximal scramblase activity. It is currently unknown if/how TMEM16F groove can open for lipid scrambling. Here we describe the analysis of ~400 µs all-atom molecular dynamics (MD) simulations of the TMEM16F revealing an allosteric mechanism leading to an open-groove, lipid scrambling competent state of the protein. The groove opens into a continuous hydrophilic conduit that is highly similar in structure to that seen in other activated scramblases. The allosteric pathway connects this opening to an observed destabilization of the Ca 2+ ion bound at the distal site near the dimer interface, to the dynamics of specific protein regions that produces the open-groove state to scramble phospholipids.