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The confinement of neuronal activity to specific subcellular regions is a mechanism for expanding the computational properties of neurons. Although the circuit organization underlying compartmentalized activity has been studied in [...]

The hallmark of the movement disorder Parkinson's disease (PD) is progressive degeneration of dopaminergic neurons. Mitochondrial dysfunction, impaired ubiquitin-mediated proteolysis of α-synuclein, and ER stress are each implicated in the complex and poorly understood sequence of events leading to dopaminergic neuron demise. In this issue of the JCI, Selvaraj et al. report that in a mouse neurotoxin-based model of PD, reduced [Ca.sup.2+] influx through transient receptor potential C1 (TRPC1) channels in the plasma membrane of dopaminergic neurons triggers a cell death-inducing ER stress response. These new findings suggest that TRPC1 channels normally function in [Ca.sup.2+]-mediated signaling pathways that couple adaptive/neurotrophic responses to metabolic and oxidative stress and suggest that disruption of these pathways may contribute to PD., Introduction Approximately one million Americans have Parkinson's disease (PD), a fatal neurodegenerative disorder that involves progressive dysfunction and death of neurons in the brain stem, midbrain, and cerebral cortex. The [...]

In skinned myocardium, cyclic AMP--dependent protein kinase A (PKA)-catalyzed phosphorylation of cardiac myosin--binding protein C (cMyBP-C) and cardiac troponin I (cTnI) is associated with a reduction in the [Ca.sup.2+] responsiveness of myofilaments and an acceleration in the kinetics of cross-bridge cycling, although the respective contribution of these two proteins remains controversial. To further examine the relative roles that cTnI and cMyBP-C phosphorylation play in altering myocardial function, we determined the [Ca.sup.2+] sensitivity of force ([pCa.sub.50]) and the activation dependence of the rate of force redevelopment ([k.sub.tr]) in control and PKA-treated mouse myocardium (isolated in the presence of 2,3-butanedione monoxime) expressing: (a) phosphorylatable cTnI and cMyBP-C (wild type [WT]), (b) phosphorylatable cTnI on a cMyBP-C--null background ([cMyBP-C.sup.-/-]), (c) nonphosphorylatable cTnI with [serines.sup.23/24/43/45] and [threonine.sup.144] mutated to alanines ([cTnI.sub.Ala5]), and (d) nonphosphorylatable cTnI on a cMyBP-C-null background ([cTnI.sub.Ala5]/[cMyBP-C.sup.-/-]). Here, PKA treatment decreased [pCa.sub.50] in WT, [cTnI.sub.Ala5], and [cMyBP-C.sup.-/-] myocardium by 0.13, 0.08, and 0.09 pCa units, respectively, but had no effect in [cTnI.sub.Ala5]/[cMyBP-C.sup.-/-] myocardium. In WT and [cTnI.sub.Ala5] myocardium, PKA treatment also increased [k.sub.tr] at submaximal levels of activation; however, PKA treatment did not have an effect on [k.sub.tr] in [cMyBP-C.sup.-/-] or [cTnI.sub.Ala5]/[cMyBP-C.sup.-/-] myocardium. In addition, reconstitution of [cTnI.sub.Ala5]/[cMyBP-C.sup.-/-] myocardium with recombinant cMyBP-C restored the effects of PKA treatment on [pCa.sub.50] and [k.sub.tr] reported in [cTnI.sub.Ala5] myocardium. Collectively, these results indicate that the attenuation in myofilament force response to PKA occurs as a result of both cTnI and cMyBP-C phosphorylation, and that the reduction in [pCa.sub.50] mediated by cMyBP-C phosphorylation most likely arises from an accelerated cross-bridge cycling kinetics partly as a result of an increased rate constant of cross-bridge detachment. 10.1085/jgp.201010448

The purpose of this study was to investigate whether diets differing in Ca concentration would have an influence on vitamin D (VitD) receptor (VDR) and calbindin D9k (Calb9k) immunoreactivities in the gastrointestinal tract of growing goats. In addition, the effect of a single VitD injection was studied, to clarify whether exogenous VitD would further increase the active Ca absorption mechanisms. The hypothesis of the study was that reduced Ca intake would lead to greater active Ca absorption, and with that, to greater amounts of VDR and Calb9k immunoreactivities. The normal Ca kid group (according to age requirements) received 2.5 to 6 g of Ca/d, whereas the lesser Ca kid group (less than requirements) received 1.5 to 4 g of Ca/d from wk 6 (weaning) to 15 (slaughter). In addition, 5 and 6 goat kids, respectively, of each group (normal Ca kid group, lesser Ca kid group), were injected with VitD (0.05 mg of cholecalciferol/kg of BW) in wk 14 of life. Blood samples were taken in wk 14 and 15. Calcium and VitD (25-hydroxyvitamin D and 1,25-dihydroxyvitamin D) concentrations were determined in serum. Immediately after slaughter, the duodenum (DD) and rumen (RU) were mounted in conventional Ussing chambers. Unidirectional flux rates of Ca across gastrointestinal tissues were measured. Additionally, tissue specimens of the gastrointestinal tract were collected, and formaldehyde-fixed paraffin sections were used for VDR and Calb9k immunohistochemistry. In all kid groups, a net absorption in the RU and a net secretion of Ca in the DD were observed. Immunoreactions of VDR were greatest in the duodenal mucosa, whereas Calb9k immunoreactions were observed in the forestomach and intestinal tissues. The greatest expression was observed in the duodenal surface epithelium. Additionally, in the VitD-injected groups, an immuno-reaction occurred in the jejunal superficial and basal glands and the ileal superficial epithelium. In contrast, the other groups showed no Calb9k immunoreactions at these sites. In conclusion, there is clear evidence for the RU as a main site for Ca absorption. The results of this study also indicate that VDR and Calb9k are highly expressed in the duodenal mucosa. The active absorption may not have such an important role in the DD because active transport was also evident in the RU. However, Calb9k expression seems to be stimulated by VitD administration. Key words: calbindin D9k, calcium, goat, vitamin D, vitamin D receptor doi: 10.2527/jas.2009-2682

Neurotransmitter release is mediated by the SNARE proteins synaptobrevin II (sybll, also known as VAMP2), syntaxin, and SNAP-25, generating a force transfer to the membranes and inducing fusion pore formation. However, the molecular mechanism by which this force leads to opening of a fusion pore remains elusive. Here we show that the ability of sybll to support exocytosis is inhibited by addition of one or two residues to the sybll C terminus depending on their energy of transfer from water to the membrane interface, following a Roltzmann distribution. These results suggest that following stimulation, the SNARE complex pulls the C terminus, of sybll deeper into the veside membrane. We propose that this movement disrupts the vesicular membrane continuity leading to fusion pore formation. In contrast to current models, the experiments suggest that fusion pore formation begins with molecular rearrangements at the intravesicular membrane leaflet and not between the apposed cytoplasmic leaflets. chromaffin cell | patch clamp capacitance measurement | caged calcium | amperometry | electrochemical detector array doi/ 10.1073/pnas.1006727107

Dietary calcium is believed to reduce colon cancer risk, but the mechanism by which this occurs is poorly understood. Employing the Citrobacter rodentium-induced transmissible murine colonic hyperplasia (TMCH) model, we previously showed that a high-calcium diet (hCa) significantly abrogated hyperplasia in the distal colons of NIH-Swiss mice. Here, we explored the mechanism of dietary protection by hCa by analyzing the expression of genes involved in the regulation of Ca uptake/flux in the intestinal epithelium, including the Ca-sensing receptor, vitamin D receptor, Ca binding protein, and transient receptor potential cation channels, subfamily V, members 5 and 6 (TRPV5/6). Interestingly, while TRPV6 expression increased significantly during TMCH, the expression of the other gene products was unchanged. This elevated TRPV6 expression was significantly abrogated by a hCa diet. Immunofluorescence revealed apical membrane localization of TRPV6 in the normal colon, whereas during TMCH we observed intense apical pole and cytoplasmic staining along the entire longitudinal crypt axis, including the expanded proliferating zone. The hCa diet reversed this effect. In humans, overexpression of TRPV6 was associated with early-stage colon cancer, and in colon carcinoma cells, inhibition of TRPV6 expression by small interfering RNA inhibited their proliferation and induced apoptosis. TRPV6 small interfering RNA also diminished the transcriptional activity of the calcium-dependent nuclear factors in activated T cells. Thus the aberrant overexpression of TRPV6 contributes to colonic crypt hyperplasia in mice and to colon cancer cell proliferation in humans. Therefore, it is likely that suppression of TRPV6 by a hCa diet is required for its protective effects in the colon. crypt hyperplasia; TRPV6; cancer prevention doi: 10.1152/ajpgi.00193.2010.

Bicarbonate secretion in the intestine (duodenum) of marine fish has been suggested to play a major role in regulation of calcium availability for uptake. However, while the end process may lead to carbonate precipitation, regulation of transport of calcium and/or bicarbonate may actually result in fine-tuning of calcium availability for transport. To test this hypothesis, sea bream (Sparus auratus) duodenal preparations were mounted in Ussing-type chambers and the effect of parathyroid hormone-related protein (PTHrP) and stanniocalcin 1 (STC 1) on the control of intestinal bicarbonate secretion and calcium transport was analyzed. As expected, PTHrP increased net calcium uptake, as a result of an increase of calcium uptake without changes in calcium efflux. In contrast, purified sea bream STC 1 caused a minor decrease of calcium uptake and a two- to threefold increase in calcium efflux. As a result, STC 1 was able to invert the calcium flux from net calcium uptake to net calcium loss, which is in keeping with its known actions as a hypocalcemic factor. Furthermore, both PTHrP and STC 1 regulate intestinal bicarbonate secretion. PTHrP increased calcium uptake and simultaneously reduced the single factor that induces calcium precipitation, bicarbonate secretion. In contrast, STC 1, while reversing the calcium net flux to make it secretory, promoted intestinal bicarbonate secretion, both actions directed to decrease the calcium gradient across the epithelium and promote immobilization in the form of bicarbonate in the intestinal lumen. Together our results provide robust evidence to support an antagonistic action of PTHrP and STC 1 in the fine control of movements of both calcium and bicarbonate in the intestine of seawater fish. ion regulation; sea water; sea bream doi: 10.1152/ajpregu.00378.2009.

We tested the hypothesis that cladocerans with higher calcium (Ca) content are more susceptible to Ca limitation by conducting life table experiments using four cladoceran species with contrasting Ca contents (0.06 2.24% of dry weight). Populations of Daphnia carinata and Daphnia galeata with high Ca content might collapse when the ambient Ca concentration is < 0.5 mg [L.sup.-1], whereas Ceriodaphnia dubia, with intermediate Ca content, and Moina macrocopa, with low Ca content, are well adapted to that low Ca level. However, Ca content is not a good proxy of the susceptibilities to Ca limitation within the genus of Daphnia. We propose an index, which considers both the Ca demand (i.e., Ca content under Ca-sufficient conditions) and the ability of cladocerans to extract and retain Ca in low-Ca environments, to explain the differences in cladoceran tolerances to Ca deficiency. We also used physiologically based biokinetic parameters, including the influx rate and efflux rate constant of Ca, to predict the interspecies differences of specific Ca content. Low-Ca species had a lower influx rate and a higher efflux rate constant of Ca than the high-Ca species. A Ca concentration of 2 mg [L.sup.-1] was sufficient to keep Daphnia spp. from extinction because of Ca limitation. doi: 10.4319/lo.2010.55.3.1426

Patients with Duchenne muscular dystrophy (DMD) have a progressive dilated cardiomyopathy associated with fatal cardiac arrhythmias. Electrical and functional abnormalities have been attributed to cardiac fibrosis; however, electrical abnormalities may occur in the absence of overt cardiac histopathology. Here we show that structural and functional remodeling of the cardiac sarcoplasmic reticulum (SR) [Ca.sup.2+] release channel/ryanodine receptor (RyR2) occurs in the mdx mouse model of DMD. RyR2 from mdx hearts were S-nitrosylated and depleted of calstabin2 (FKBP12.6), resulting in 'leaky' RyR2 channels and a diastolic SR [Ca.sup.2+] leak. Inhibiting the depletion of calstabin2 from the RyR2 complex with the [Ca.sup.2+] channel stabilizer S107 ('rycal') inhibited the SR [Ca.sup.2+] leak, inhibited aberrant depolarization in isolated cardiomyocytes, and prevented arrhythmias in vivo. This suggests that diastolic SR [Ca.sup.2+] leak via RyR2 due to S-nitrosylation of the channel and calstabin2 depletion from the channel complex likely triggers cardiac arrhythmias. Normalization of the RyR2-mediated diastolic SR [Ca.sup.2+] leak prevents fatal sudden cardiac arrhythmias in DMD. calcium | excitation-contraction coupling | heart | sudden cardiac death | myopathy doi/ 10.1073/pnas.0908540107

Problem statement: Defining of correlation between toxic and essential elements in human beings is an important clinical screening procedure. Approach: The aim of this study was to determine the correlation between cadmium as toxic element and calcium as an essential element in the blood at the smokers. We have investigated 50 human blood samples of different ages and genders (age: 35-45 years old), of the citizens from the Municipality of Dragash (an environment without pollution). Results: The results that were achieved in this study showed the significant difference in the average of cadmium concentration in human blood of the smokers group from nonsmokers group (p = Conclusion/Recommendations: Correlation between [Cd.sup.2+] and [Ca.sup.2+] in the human blood with a high statistical significance in the group of smokers comes as a result of powerful competitive reaction between [Cd.sup.2+] and [Ca.sup.2+] in biochemical processes. Competitive reaction between [Cd.sup.2+] and [Ca.sup.2+] in biochemical processes. Key words: Cadmium, calcium, smoking, blood, INTRODUCTION Cadmium is one of the most commonly found toxic metals present in our environment. The major route of exposure to cadmium for the general non-smoking population is via food, [...]

Muscular dystrophy is a general term encompassing muscle disorders that cause weakness and wasting, typically leading to premature death. Membrane instability, as a result of a genetic disruption within the dystrophin-glycoprotein complex (DGC), is thought to induce myofiber degeneration, although the downstream mechanism whereby membrane fragility leads to disease remains controversial. One potential mechanism that has yet to be definitively proven in vivo is that unregulated calcium influx initiates disease in dystrophic myofibers. Here we demonstrate that calcium itself is sufficient to cause a dystrophic phenotype in skeletal muscle independent of membrane fragility. For example, overexpression of transient receptor potential canonical 3 (TRPC3) and the associated increase in calcium influx resulted in a phenotype of muscular dystrophy nearly identical to that observed in DGC-lacking dystrophic disease models, including a highly similar molecular signature of gene expression changes. Furthermore, transgene-mediated inhibition of TRPC channels in mice dramatically reduced calcium influx and dystrophic disease manifestations associated with the mdx mutation (dystrophin gene) and deletion of the [delta]-sarcoglycan (Scgd) gene. These results demonstrate that calcium itself is sufficient to induce muscular dystrophy in vivo, and that TRPC channels are key disease initiators downstream of the unstable membrane that characterizes many types of muscular dystrophy. fibrosis | necrosis | degeneration | skeletal muscle www.pnas.org/cgi/doi/10.1073/pnas.0906591106

Calcium (Ca) supplements, especially Ca carbonate (CaC[O.sub.3]), are the main alternative sources of dietary Ca and an important part of a treatment regimen for osteoporosis, the most common metabolic bone disorder of aging and menopause. In a female ovariectomized (OVX) rat model for studying postmenopausal osteoporosis, we tested the hypothesis that a small compared with a large particle size of GAG[O.sub.3] (13.0- vs. 18.5-[micro]m geometric diameter) would result in increased Ca balance and subsequently bone mass and that this would be affected by dietary Ca level. We used 6-moold rats that were OVX either at 6 or 3 mo of age as models of early or stable menopausal status, respectively. The rats received semipurified diets that contained either 0.4 or 0.2% dietary Ca provided from CaC[O.sub.3] of 2 particle sizes. A group of Sham-operated rats with intact ovaries served as control and were fed 0.4% dietary Ca from large particles. Estrogen deficiency as a result of ovariectomy had an adverse effect on bone density, mineral content, and bone mechanical properties (P < 0.001 ). Reducing dietary Ca from 0.4 to 0.2% resulted in significant adverse effects on bone density and mechanical properties (P < 0.001). The particle size of CaC[O.sub.3] did not affect total Ca balance, bone dual energy X-ray absorptiometry and peripheral quantitative computed tomography indices, bone ash and Ca content, or the mechanical determinants of bone strength. We conclude that a decrease in particle size of CaC[O.sub.3] to 70% of that typically found in Ca supplements does not provide a benefit to overall Ca metabolism or bone characteristics and that the amount of Ca consumed is of greater influence in enhancing Ca nutrition and skeletal strength.

Calcium has been proposed as a primary influence on cell death during ischemic episodes in myocardial cells. One component of calcium entry into a cell is resting calcium influx. This basal movement of calcium is blocked by 100 µmol/L gadolinium chloride (Gd[Cl.sub.3]) in cardiac myocytes. Therefore, Gd[Cl.sub.3] should be cardioprotective under anoxic conditions. To test this, cardiac myocytes isolated from adult male rats were subjected to anoxia (100% [N.sub.2]) in the presence or absence of 100 µmol/L Gd[Cl.sub.3] in one of 2 ways. In the first method, cells were suspended in media and rendered anoxic for 0, 30, and 60 min, after which cell morphology and viability were scored. After 60 min of anoxia, rod-shaped cells accounted for 46% ± 4% of total cells (viability 81%); 10 min of reoxygenation markedly reduced rod-shaped cells to 27% (viability 72%). Gd[Cl.sub.3] in the medium did not protect the cells (anoxic rods 49%, reoxygenated rods 30%, viability 77%). In the second method, cells were attached to a laminin substrate, rendered anoxic, and then videotaped for up to 6 h. In this system, cells maintained their shape for some time after the onset of anoxia, and then began to 'die' (i.e., to take on either a rigor form or hypercontracted form) at a measurable rate. Time to onset of 'death' ([t.sub.0]), time to 50% and 100% 'death' ([t.sub.50] and [t.sub.100]), and rate of 'death' were used to measure anoxic damage. Without Gd[Cl.sub.3], cells on average began to die 115 ± 32 min after the onset of anoxia ([t.sub.0]); they died at an average rate of 0.046 cells/min. [t.sub.50] was achieved in 149 ± 42 min, [t.sub.100] in 183 ± 54 min. Addition of 100 µmol/L Gd[Cl.sub.3] did not affect any of these parameters. We concluded that Gd[Cl.sub.3] was not cardioprotective for anoxic myocytes and that cell damage generated by anoxia could not be attributed to resting calcium influx. Key words: heart, myocyte, calcium, anoxia. Le calcium aurait une influence imporante sur la mort cellulaire durant des episodes d'ischemie dans les cellules myocardiques. Une composante de l'entree de calcium dans une cellule est le courant calcique entrant au repos. Ce mouvement basal de calcium est bloque par 100 [micron]mol/L de chlorure de gadolinium (Gd[Cl.sub.3]) dans les myocytes cardiaques. Par consequent, le Gd[Cl.sub.3] devrait avoir un effet cardioprotecteur dans des conditions anoxiques. Pour verifier cette hypothese, on a soumis des cardiomyocytes isoles de rats males adultes a une anoxie (100% [N.sub.2]) en presence ou en l'absence de 100 [micron]mol/L de Gd[Cl.sub.3], en utilisant deux methodes. Dans la premiere methode, les cellules ont ete suspendues dans des solutions et rendues anoxiques pendant 0, 30, et 60 min; la morphologie et la viabilite des cellules ont ensuite ete notees. Apres 60 min d'anoxie, les cellules en batonnet ont represente 46% [+ o -] 4% de la totalite des cellules (viabilite 81%); une reoxygenation pendant 10 min a reduit de facon marquee le nombre de cellules en batonnet a 27% (viabilite 72%). La presence de Gd[Cl.sub.3] dans la solution n'a pas protege les cellules (batonnets anoxiques 49%, batonnets reox. 30%, viabilite 77%). Dans la seconde methode, les cellules ont ete fixees sur un substrat de laminine, rendues anoxiques, puis leur comportement a ete enregistre sur bande video pendant une periode allant jusqu'a 6 h. Dans ce systeme, les cellules ont maintenu leur forme quelque temps apres le debut de l'anoxie, puis ont commence a << mourir >> (c.-a-d. a prendre une forme soit rigide, soit hypercontractee) a un taux mesurable. Le temps vers le debut de la << mort >> [t.sub.0], le temps vers une << mortalite >> de 50% et de 100% ([t.sub.50] et [t.sub.100]) et le taux de << mortalite >> ont ete utilises pour mesurer la lesion anoxique. Sans Gd[Cl.sub.3], les cellules en moyenne ont commence a << mourir >> 115 [+ o -] 32 min apres le declenchement de l'anoxie ([t.sub.0]); les cellules sont << mortes >> a un taux moyen de 0,046 cellules/min. [t.sub.50] a ete atteint en 149 [+ o -] 42 min, [t.sub.100] en 183 [+ o -] 54 min. L'ajout de 100 [micron]mol/L de Gd[Cl.sub.3] n'a influe sur aucun de ces parametres. Nous avons conclu que le Gd[Cl.sub.3] n'a pas eu d'effet cardioprotecteur sur les myocytes anoxiques et que l'alteration des cellules causee par l'anoxie ne pouvait etre attribue e a un courant entant de calcium au repos. Mots-cles : coeur, myocyte, calcium, anoxie. [Traduit par la Redaction], Introduction Loss of control of calcium pathways is one of the features of reoxygenation after myocardial ischemia in mammals. In the cardiac myocyte, voltage-gated calcium channels, stretch-activated channels, and background [...]

Endurance impact exercise, e.g., running, is known to enhance the intestinal calcium absorption. However, nonimpact exercise, e.g., swimming, is more appropriate for osteoporotic patients with cardiovascular diseases or disorders of bone and joint, but the effect of swimming on the intestinal calcium transport was unknown. This study, therefore, aimed to investigate the transepithelial calcium transport and the expression of related genes in the intestine of rats trained to swim nonstop 1 h/day, 5 days/wk for 2 wk. We found that endurance swimming stimulated calcium transport in the duodenum, proximal jejunum, and cecum, while decreasing that in the proximal colon. Swimming affected neither the transepithelial potential difference nor resistance. As demonstrated by real-time PCR, the small intestine, especially the duodenum, responded to swimming by upregulating a number of genes related to the transcellular calcium transport, i.e., TRPV5, TRPV6, calbindin-[D.sub.9k], PMC[A.sub.1b], and NCX1, and the paracellular calcium transport, i.e., ZO-1, ZO-2, ZO-3, cingulin, occludin, and claudins, as well as nuclear receptor of 1,25[(OH).sub.2][D.sub.3]. In contrast, swimming downregulated those genes in the colon. Microarray analysis showed that swimming also altered the expression of duodenal genes related to the transport of several ions and nutrients, e.g., [Na.sup.+], [K.sup.+], [C1.sup.-], glucose, and amino acids. In conclusion, endurance swimming enhanced intestinal calcium absorption, in part, by upregulating the calcium transporter genes. The present microarray study also provided relevant information for further investigations into the intestinal nutrient and electrolyte transport during nonimpact exercise. gene ontology; microarray; nonimpact exercise; real-time polymerase chain reaction; Ussing chamber

There are strong links between obesity, elevated free fatty acids, and type 2 diabetes. Specifically, the saturated fatty acid palmitate has pleiotropic effects on [beta]-cell function and survival. In the present study, we sought to determine the mechanism by which palmitate affects intracellular [Ca.sup.2+], and in particular the role of the endoplasmic reticulum (ER). In human [beta]-cells and MIN6 cells, palmitate rapidly increased cytosolic [Ca.sup.2+] through a combination of [Ca.sup.2+] store release and extracellular [Ca.sup.2+] influx. Palmitate caused a reversible lowering of ER [Ca.sup.2+], measured directly with the fluorescent protein-based ER [Ca.sup.2+] sensor D1ER. Using another genetically encoded indicator, we observed long-lasting oscillations of cytosolic [Ca.sup.2+] in palmitate-treated cells. In keeping with this observed ER [Ca.sup.2+] depletion, palmitate induced rapid phosphorylation of the ER [Ca.sup.2+] sensor protein kinase R-like ER kinase (PERK) and subsequently ER stress and [beta]-cell death. We detected little palmitate-induced insulin secretion, suggesting that these [Ca.sup.2+] signals are poorly coupled to exocytosis. In summary, we have characterized [Ca.sup.2+]-dependent mechanisms involved in altered [beta]-cell function and survival induced by the free fatty acid palmitate. We present the first direct evidence that free fatty acids reduce ER [Ca.sup.2+] and shed light on pathways involved in lipotoxicity and the pathogenesis of type 2 diabetes. diabetes; free fatty acids; fluorescence resonance energy transfer; calcium homeostasis; endoplasmic reticulum

Phospholemman (PLM) belongs to the FXYD family of small ion transport regulators. When phosphorylated at [Ser.sup.68], PLM inhibits cardiac [Na.sup.+]/[Ca.sup.2+] exchanger (NCX1). We previously demonstrated that the cytoplasmic tail of PLM interacts with the proximal intracellular loop (residues 218-358), but not the transmembrane (residues 1-217 and 765-938) or [Ca.sup.2+]-binding (residues 371-508) domains, of NCX1. In this study, we used intact [Na.sup.+]/[Ca.sup.2+] exchanger with various deletions in the intracellular loop to map the interaction sites with PLM. We first demonstrated by Western blotting and confocal immunofluorescence microscopy that wild-type (WT) NCX1 and its deletion mutants were expressed in transfected HEK-293 cells. Cotransfection with PLM and NCX1 (or its deletion mutants) in HEK-293 cells did not decrease expression of NCX1 (or its deletion mutants). Coexpression of PLM with WT NCX1 inhibited NCX1 current ([I.sub.NaCa]). Deletion of residues 240-679, 265-373, 250-300, or 300-373 from WT NCX1 resulted in loss of inhibition of [I.sub.NaCa] by PLM. Inhibition of [I.sub.NaCa] by PLM was preserved when residues 229-237, 270-300, 328-330, or 330-373 were deleted from the intracellular loop of NCX1. These results suggest that PLM mediated inhibition of [I.sub.NaCa] by interacting with two distinct regions (residues 238-270 and 300-328) of NCX1. Indeed, [I.sub.NaCa] measured in mutants lacking residues 238-270, 300-328, or 238270 + 300-328 was not affected by PLM. Ghitathione S-transferase pull-down assays confirmed that PLM bound to fragments corresponding to residues 218-371, 218-320, 218-270, 238-371, and 300-373, but not to fragments encompassing residues 250-300 and 371-508 of NCX1, indicating that residues 218-270 and 300-373 physically associated with PLM. Finally, acute regulation of [I.sub.NaCa] by PLM phosphorylation observed with WT NCX1 was absent in 250300 deletion mutant but preserved in 229-237 deletion mutant. We conclude that PLM mediates its inhibition of NCX1 by interacting with residues 238-270 and 300-328. FXYD1; ion transport; sodium-calcium exchange

When skeletal muscle is stretched or injured, myogenic satellite cells are activated to enter the cell cycle. This process depends on nitric oxide (NO) production by NO synthase (NOS), matrix metalloproteinase activation, release of hepatocyte growth factor (HGF) from the extraeellular matrix, and presentation of HGF to the c-met receptor as demonstrated by a primary culture and in vivo assays. We now add evidence that calcium-calmodulin is involved in the satellite cell activation cascade in vitro. Conditioned medium from cultures that were treated with a calcium ionophore (A23187, ionomycin) for 2 h activated cultured satellite cells and contained active HGF, similar to the effect of mechanical stretch or NO donor treatments. The response was abolished by addition of calmodulin inhibitors (calmidazolium, W-13, W-12) or a NOS inhibitor [N.sup.G]-nitro-L-arginine methyl ester hydrochloride but not by its less inactive enantiomer [N.sup.G]-nitro-Darginine methyl ester hydrochloride. Satellite cells were also shown to express functional calmodulin protein having a calcium-binding activity at 12 h postplating, which is the time at which the calcium ionophore was added in this study and the stretch treatment was applied in our previous experiments. Therefore, results from these experiments provide an additional insight that calcium-calmodulin mediates HGF release from the matrix and that this step in the activation pathway is upstream from NO synthesis. muscle regeneration; stretch-activation

It has been suggested that store-operated [Ca.sup.2+] entry (SOC) facilitates catecholamine secretion and synthesis in bovine adrenal medullary (AM) cells. However, there has been no experimental result clearly showing that cation channel activity is enhanced by store [Ca.sup.2+] depletion. Thus the present experiments were undertaken to address the issue of whether rat AM cells have SOC channels. Inhibition of the sarco(endo)plasmic reticulum [Ca.sup.2+] (SERCA) pump resulted in a sustained increase in intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]) in rat AM cells. This increase was completely suppressed by 2 mM [Ni.sup.2+] but not by 100 [micro]M D600. A bath application of [Ni.sup.2+], but not D600, produced an outward current at -60 mV in rat AM cells, whereas exposure to a SERCA pump inhibitor did not affect either the whole cell current level or the [Ni.sup.2+]-induced outward current. The refilling of intracellular store sites was suppressed by the addition of [Ni.sup.2+] to the perfusate. RT-PCR revealed that transcripts for transient receptor potential channels 1 (TRPC1) and 5 (TRPC5) were present in rat adrenal medullas. Immunocytochemistry showed that TRPC1 channels, which have been implicated in SOC in certain types of cells, were mainly localized in the endoplasmic reticulum (ER) and not in the plasma membrane, and that STIM1, a [Ca.sup.2+] sensor in the ER, was not expressed in rat AM cells. On the basis of these results, we conclude that rat AM cells lack the SOC mechanism. calcium store sites; transient receptor potential channel; endoplasmic reticulum

Relatively little is known about the contribution of [Ca.sup.2+]-dependent and -independent mechanisms in the contractility of neonatal gastrointestinal smooth muscle. We therefore studied [Ca.sup.2+] homeostasis and [Ca.sup.2+] sensitization mechanisms in 10-day-old and adult guinea pig gallbladder smooth muscle to elucidate developmental changes in these processes. Gallbladder contractility was evaluated by isometrical tension recordings from strips, intracellular [Ca.sup.2+] concentration was estimated by epifluorescence microscopy of fura-2-loaded isolated cells, and protein expression and phosphorylation were assessed by Western blot analysis. The neonatal gallbladder contracted significantly less to CCK than adult tissue, but this correlated with an increased [Ca.sup.2+] mobilization, suggesting immaturity of [Ca.sup.2+] sensitization mechanisms. The enhanced [Ca.sup.2+] release in the newborn gallbladder was the result of the increase in the size of the releasable [Ca.sup.2+] pool. Moreover, in neonatal smooth muscle cells, neither the plasma membrane [Ca.sup.2+] pump nor the [Na.sup.+]/[Ca.sup.2+] exchanger collaborate in the extrusion of [Ca.sup.2+]. In contrast, in these cells, there is an increase in phospholamban phosphorylation, which could drive to an overactivity of the sarco(endo)plasmic reticulum [Ca.sup.2+]-ATPase pump. The reduced [Ca.sup.2+] sensitivity in neonatal tissues was demonstrated by the lack of effect to Y-27362, an inhibitor of Rho kinase (ROCK), and GF-109203X, an inhibitor of PKC, on agonist-induced contraction. In addition, the neonatal gallbladder showed lower levels of RhoA, ROCK, PKC, and two effectors [C-kinase-dependent inhibitor of 17 kDa (CPI-17) and myosin phosphatase targetting 1 (MYPT1)] as well as an absence of CPI-17 and MYPT1 phosphorylation in response to agonists. In conclusion, our results indicate that the main mechanisms involved in smooth muscle contractility are under developmental regulation. [Ca.sup.2+]-dependent contraction; [Ca.sup.2+] sensitization; gastrointestinal smooth muscle

We hypothesized that testosterone at physiological levels enhances cardiac contractile responses to stimulation of both [[alpha].sub.1]- and [[beta].sub.1]-adrenoceptors by increasing [Ca.sup.2+] release from the sarcoplasmic reticulum (SR) and speedier removal of [Ca.sup.2+] from cytosol via [Ca.sup.2+]-regulatory proteins. We first determined the left ventricular developed pressure, velocity of contraction and relaxation, and heart rate in perfused hearts isolated from control rats, orchiectomized rats, and orchiectomized rats without and with testosterone replacement (200 [micro]g/100 g body wt) in the presence of norepinephrine ([10.sup.-7] M), the [[alpha].sub.1]-adrenoceptor agonist phenylephrine ([10.sup.-6] M), or the nonselective [beta]-adrenoceptor agonist isoprenaline ([10.sup.-7] M) in the presence of 5 x [10.sup.-7] M ICI-118,551, a [[beta].sub.2]-adrenoceptor antagonist. Next, we determined the amplitudes of intracellular [Ca.sup.2+] concentration transients induced by electrical stimulation or caffeine, which represent, respectively, [Ca.sup.2+] release via the ryanodine receptor (RyR) or releasable [Ca.sup.2+] in the SR, in ventricular myocytes isolated from the three groups of rats. We also measured [sup.45][Ca.sup.2+] release via the RyR. We then determined the time to 50% decay of both transients, which represents, respectively, [Ca.sup.2+] reuptake by sarco(endo)plasmic reticulum [Ca.sup.2+]-ATPase (SERCA) and removal via the sarcolemmal [Na.sup.+]/[Ca.sup.2+] exchanger (NCX). We correlated [Ca.sup.2+] removal from the cytosol with activities of SERCA and its regulator phospholamban as well as NCX. The results showed that testosterone at physiological levels enhanced positive inotropic and lusitropic responses to stimulation of [[alpha].sub.1]- and [[beta].sub.1]-adrenoceptors via the androgen receptor. The increased contractility and speedier relaxation were associated with increased [Ca.sup.2+] release via the RyR and faster [Ca.sup.2+] removal out of the cytosol via SERCA and NCX. orchiectomy; androgen receptor; left ventricular developed pressure; velocity of contraction and relaxation; electrical-induced intracellular [Ca.sup.2+] concentration transients; caffeine-induced intracellular [Ca.sup.2+] concentration transients; ryanodine receptor; sarco(endo)plasmic reticulum [Ca.sup.2+]-ATPase; [Na.sup.+]/[Ca.sup.2+] exchanger

In smooth muscle, the gating of dihydropyridine-sensitive [Ca.sup.2+] channels may either be stochastic and voltage dependent or coordinated among channels and constitutively active. Each form of gating has been proposed to be largely responsible for [Ca.sup.2+] influx and determining the bulk average cytoplasmic Ca concentration. Here, the contribution of voltage-dependent and constitutively active channel behavior to [Ca.sup.2+] signaling has been studied in voltage-clamped single vascular and gastrointestinal smooth muscle cells using wide-field epifluorescence with near simultaneous total internal reflection fluorescence microscopy. Depolarization (-70 to +10 mV) activated a dihydropyridine-sensitive voltage-dependent [Ca.sup.2+] current ([I.sub.Ca]) and evoked a rise in [[Ca.sup.2+]] in each of the subplasma membrane space and bulk cytoplasm. In various regions of the bulk cytoplasm the [[Ca.sup.2+]] increase ([[[Ca.sup.2+]].sub.c]) was approximately uniform, whereas that of the subplasma membrane space ([[[Ca.sup.2+]].sub.PM]) had a wide range of amplitudes and time courses. The variations that occurred in the subplasma membrane space presumably reflected an uneven distribution of active [Ca.sup.2+] channels (clusters) across the sarcolemma, and their activation appeared consistent with normal voltage-dependent behavior. Indeed, in the present study, dihydropyridine-sensitive [Ca.sup.2+] channels were not normally constitutively active. The repetitive localized [[[Ca.sup.2+]].sub.PM] rises ('persistent [Ca.sup.2+] sparklets') that characterize constitutively active channels were observed rarely (2 of 306 cells). Neither did dihydropyridine-sensitive constitutively active [Ca.sup.2+] channels regulate the bulk average [[[Ca.sup.2+]].sub.c], A dihydropyridine blocker of [Ca.sup.2+] channels, nimodipine, which blocked [I.sub.Ca] and accompanying [[[Ca.sup.2+]].sub.c] rise, reduced neither the resting bulk average [[[Ca.sup.2+]].sub.c] (at -70 mV) nor the rise in [[[Ca.sup.2+]].sub.c], which accompanied an increased electrochemical driving force on the ion by hyperpolarization (-130 mV). Activation of protein kinase C with indolactam-V did not induce constitutive channel activity. Thus, although voltage-dependent [Ca.sup.2+] channels appear clustered in certain regions of the plasma membrane, constitutive activity is unlikely to play a major role in [[[Ca.sup.2+]].sub.c] regulation. The stochastic, voltage-dependent activity of the channel provides the major mechanism to generate rises in [[Ca.sup.2+]].

Canonical transient receptor potential (TRPC) channels control influxes of [Ca.sup.2+] and other cations that induce diverse cellular processes upon stimulation of plasma membrane receptors coupled to phospholipase C (PLC). Invention of subtype-specific inhibitors for TRPCs is crucial for distinction of respective TRPC channels that play particular physiological roles in native systems. Here, we identify a pyrazole compound (Pyr3), which selectively inhibits TRPC3 channels. Structure-function relationship studies of pyrazole compounds showed that the trichloroacrylic amide group is important for the TRPC3 selectivity of Pyr3. Electrophysiological and photoaffinity labeling experiments reveal a direct action of Pyr3 on the TRPC3 protein. In DT40 B lymphocytes, Pyr3 potently eliminated the [Ca.sup.2+] influx-dependent PLC translocation to the plasma membrane and late oscillatory phase of B cell receptor-induced [Ca.sup.2+] response. Moreover, Pyr3 attenuated activation of nuclear factor of activated T cells, a [Ca.sup.2+]-dependent transcription factor, and hypertrophic growth in rat neonatal cardiomyocytes, and in vivo pressure overload-induced cardiac hypertrophy in mice. These findings on important roles of native TRPC3 channels are strikingly consistent with previous genetic studies. Thus, the TRPC3-selective inhibitor Pyr3 is a powerful tool to study in vivo function of TRPC3, suggesting a pharmaceutical potential of Pyr3 in treatments of TRPC3-related diseases such as cardiac hypertrophy. [Ca.sup.2+] signaling | pyrazole compounds | TRPC channels | TRPC3

PDGF B chain or PDGF receptor (PDGFR)-[beta]-deficient (-/-) mice lack mesangial cells. To study responses of [alpha]- and [beta]-receptor activation to PDGF ligands, metanephric mesenchymal cells (MMCs) were established from embryonic day E11.5 wild-type (+/+) and -/- mouse embryos. PDGF BB stimulated cell migration in +/+ cells, whereas PDGF AA did not. Conversely, PDGF AA was chemotactic for -/- MMCs. The mechanism by which PDGFR-[beta] inhibited AA-induced migration was investigated. PDGF BB, but not PDGF AA, increased intracellular [Ca.sup.2+] and the production of reactive oxygen species (ROS) in +/+ cells. Transfection of -/- MMCs with the wild-type [beta]-receptor restored cell migration and ROS generation in response to PDGF BB and inhibited AA-induced migration. Inhibition of [Ca.sup.2+] signaling facilitated PDGF AA-induced chemotaxis in the wild-type cells. The antioxidant N-acetyl-L-cysteine (NAC) or the NADPH oxidase inhibitor diphenyleneiodonium (DPI) abolished the BB-induced increase in intracellular [Ca.sup.2+] concentration, suggesting that ROS act as upstream mediators of [Ca.sup.2+] in suppressing PDGF AA-induced migration. These data indicate that ROS and [Ca.sup.2+] generated by active PDGFR-[beta] play an essential role in suppressing PDGF AA-induced migration in +/+ MMCs During kidney development, PDGFR [beta]-mediated ROS generation and [Ca.sup.2+] influx suppress PDGF AA-induced chemotaxis in metanephric mesenchyme. reactive oxygen species; calcium

Calcium absorption proceeds by transcellular and paracellular flux, with the latter accounting for most absorbed calcium when calcium intake is adequate. Vitamin D helps regulate transcellular calcium transport by increasing calcium uptake via a luminal calcium channel and by inducing the cytosolic calcium transporting protein, calbindin[D.sub.9k]. Recent studies utilizing knockout mice have challenged the functional importance of the channel and calbindin. To integrate the new findings with many previous studies, the function of the two molecules must be evaluated in the calcium transport and economy of mice. When calcium intake is high, transcellular calcium transport contributes little to total calcium absorption. Therefore, increasing calcium intake seems the most effective nutritional approach to ensure adequate absorption and prevent bone loss.

Upon toll-like receptor 4 (TLR4) signaling in macrophages, the mammalian Swi/Snf-like BAF chromatin remodeling complex is recruited to many TLR4 target genes where it remodels their chromatin to promote transcription. Here, we show that, surprisingly, recruitment is not sufficient for chromatin remodeling; a second event, dependent on calcium/calmodulin (CAM), is additionally required. Calcium/CaM directly binds the HMG domain of the BAF57 subunit within the BAF complex. Calcium/CaM antagonists, including a CaM-binding peptide derived from BAF57, abolish BAF-dependent remodeling and gene expression without compromising BAF recruitment. BAF57 RNAi and BAF57 dominant negative mutants defective in CaM binding similarly impair the induction of BAF target genes. Our data implicate calcium/CaM in TLR4 signaling, and reveal a previously undescribed, recruitment-independent mode of regulation of the BAF complex that is probably achieved through a direct CaMoBAF interaction. innate immunity | Brg1 | macrophages

Small conductance [Ca.sup.2+]-activated [K.sup.+] (SK) channels sense intracellular [Ca.sup.2+] concentrations via the associated [Ca.sup.2+]-binding protein calmodulin. Structural and functional studies have revealed essential properties of the interaction between calmodulin and SK channels. However, it is not fully understood how the binding of [Ca.sup.2+] to calmodulin leads to channel opening. Drawing on previous biochemical studies of free calmodulin using lanthanide ions as [Ca.sup.2+] substitutes, we have used the lanthanide ion, [Tb.sup.3+], as an alternative ligand to study the activation properties of SK channels. We found that SK channels can be fully activated by nanomolar concentrations of [Tb.sup.3+], indicating an apparent affinity >100-fold higher than [Ca.sup.2+]. Competition experiments show that [Tb.sup.3+] binds to the same sites as [Ca.sup.2+] to activate the channels. Additionally, SK channels activated by [Tb.sup.3+] demonstrate a remarkably slow deactivation process. Comparison of our results with previous biochemical studies suggests that in the intact SK channel complex, the N-lobe of calmodulin provides ligand-binding sites for channel gating, and that its ligand-binding properties are comparable to those of the N-lobe in isolated calmodulin. lanthanide | EF hand | gating | calcium-activated potassium channel

Caenorhabditis elegans navigates thermal gradients by using a behavioral strategy that is regulated by a memory of its cultivation temperature (Tc). At temperatures above or around the Tc, animals respond to temperature changes by modulating the rate of stochastic reorientation events. The bilateral AFD neurons have been implicated as thermosensory neurons, but additional thermosensory neurons are also predicted to play a role in regulating thermotactic behaviors. Here, we show that the AWC olfactory neurons respond to temperature. Unlike AFD neurons, which respond to thermal stimuli with continuous, graded calcium signals, AWC neurons exhibit stochastic calcium events whose frequency is stimulus-correlated in a To-dependent manner. Animals lacking the AWC neurons or with hyperactive AWC neurons exhibit defects in the regulation of reorientation rate in thermotactic behavior. Our observations suggest that the AFD and AWC neurons encode thermal stimuli via distinct strategies to regulate C. elegans thermotactic behavior. calcium imaging | G protein-coupled receptor AWC neuron | isothermal tracking

To gain insight into the mechanisms that underlie angiotensin II (ANG II)-induced cytoplasmic [Ca.sup.2+] concentration ([[Ca].sub.cyt]) oscillations in medullary pericytes, we expanded a prior model of ion fluxes. ANG II stimulation was simulated by doubling maximal inositol trisphosphate (I[P.sub.3]) production and imposing a 90% blockade of [K.sup.+] channels. We investigated two configurations, one in which ryanodine receptors (RyR) and I[P.sub.3] receptors (I[P.sub.3]R) occupy a common store and a second in which they reside on separate stores. Our results suggest that [Ca.sup.2+] release from stores and import from the extracellular space are key determinants of oscillations because both raise [Ca] in subplasmalemmal spaces near RyR. When the [Ca.sup.2+]-induced [Ca.sup.2+] release (CICR) threshold of RyR is exceeded, the ensuing [Ca.sup.2+] release is limited by [Ca.sup.2+] reuptake into stores and export across the plasmalemma. If sarco(endo)plasmic reticulum [Ca.sup.2+]-ATPase (SERCA) pumps do not remain saturated and sarcoplasmic reticulum [Ca.sup.2+] stores are replenished, that phase is followed by a resumption of leak from internal stores that leads either to [[Ca].sub.cyt] elevation below the CICR threshold (no oscillations) or to elevation above it (oscillations). Our model predicts that oscillations are more prone to occur when I[P.sub.3]R and RyR stores are separate because, in that case, [Ca.sup.2+] released by RyR during CICR can enhance filling of adjacent I[P.sub.3] stores to favor a high subsequent leak that generates further CICR events. Moreover, the existence or absence of oscillations depends on the set points of several parameters, so that biological variation might well explain the presence or absence of oscillations in individual pericytes. mathematical model; sarcoplasmic reticulum stores; ryanodine receptors; inositol trisphosphate receptors; medullary pericytes

A new method for the determination of free calcium concentration in human plasma was developed by online coupling capillary electrophoresis (CE) with inductively coupled plasma optical emission spectrometry (ICP-OES). Baseline separation of calcium-containing species was achieved by CE-ICP-OES in a 120-cm-long capillary with 100-[micro]m internal diameter, at 20 kV applied voltage, with a 30 mmol/L Tris-HCl buffer at pH 7.4. A total of eight calcium-containing species were found in human plasma; the concentration of free calcium ion was found to be 41.9 mg/L. The concentrations of calcium for other seven calcium species, estimated from the calibration against [Ca.sup.2[+ or -] ] standard, were 3.14-15.6 mg/L. The precision (RSD, n = 10) ranged from 1.2 to 2.7% for the migration time and 2.8 to 3.9% for the peak area. The developed method was also applied to analyze plasma samples with recovery ranged from 94.5 to 102% for samples spiked with 40 mg/L free [Ca.sup.2[+ or -] ] ion.

The objective of this trial was to determine how 25-hydroxyvitamin [D.sub.3] (25-OH [D.sub.3]) supplementation, altering supplemental dietary calcium, or their combination influence postmortem biochemical and tenderness changes in muscles from the round of mature cows. Twenty-seven Angus cows (3 to 7 yr old) were allotted randomly to 9 pens with 3 cows per pen. Treatments were arranged in a 3 x 3 factorial design with 3 dosages of 25-OH [D.sub.3] (0, 250, or 500 mg of 25-OH [D.sub.3] administered as a 1-time oral bolus 7 d before slaughter) and 3 percentages of supplemental limestone (0.5, 0.75, and 1.0%) replenished in the diet for 3 d before slaughter and after a 2-wk limestone withdrawal. Plasma samples were obtained during the feeding period. Upon slaughter, adductor, gracilus, pectineus, sartorius, semimembranosus, vastus intermedius, and vastus lateralis muscles were obtained and aged for 1, 3, or 7 d. Calcium concentrations were increased in plasma when 250 or 500 mg of 25-OH [D.sub.3] were administered (P [less than or equal to] 0.05). Calcium concentrations in muscle increased (P [less than or equal to] 0.001) when 500 mg of 25-OH [D.sub.3] were administered. Concentrations of 25-OH [D.sub.3] in meat and in plasma and 1,25-dihydroxyvitamin [D.sub.3] [1,25-[(OH).sub.2] [D.sub.3]] in plasma were increased when 25-OH [D.sub.3] was administered (P [less than or equal to] 0.05). The percentage of limestone replenished in the diet had no effect on 25-OH [D.sub.3] or 1,25-[(OH).sub.2] [D.sub.3] in meat or in plasma. Calpastatin activity was affected by treatments only in the gracilus and vastus intermedius muscles (P [less than or equal to] 0.05). Among all muscles and aging periods, calpastatin activity and intensity of troponin-T degradation product were related inversely. Results indicate that supplemental 25-OH [D.sub.3] has some influence on muscle characteristics known to improve tenderness, but improved tenderness was not observed. Key words: beef, calcium, cow, 25-hydroxyvitamin [D.sub.3], tenderness

Head-to-tail polymerization of tropomyosin is crucial for its actin binding, function in actin filament assembly, and the regulation of actin-myosin contraction. Here, we describe the 2.1 A resolution structure of crystals containing overlapping tropomyosin N and C termini (TM-N and TM-C) and the 2.9 A resolution structure of crystals containing TM-N and TM-C together with a fragment of troponin-T (TnT). At each junction, the N-terminal helices of TM-N were splayed, with only one of them packing against TM-C. In the C-terminal region of TM-C, a crucial water in the coiled-coil core broke the local 2-fold symmetry and helps generate a kink on one helix. In the presence of a TnT fragment, the asymmetry in TM-C facilitates formation of a 4-helix bundle containing two TM-C chains and one chain each of TM-N and TnT. Mutating the residues that generate the asymmetry in TM-C caused a marked decrease in the affinity of troponin for actin-tropomyosin filaments. The highly conserved region of TnT, in which most cardiomyopathy mutations reside, is crucial for interacting with tropomyosin. The structure of the ternary complex also explains why the skeletal- and cardiac-muscle specific C-terminal region is required to bind TnT and why tropomyosin homodimers bind only a single TnT. On actin filaments, the head-to-tail junction can function as a molecular swivel to accommodate irregularities in the coiled-coil path between successive tropomyosins enabling each to interact equivalently with the actin helix. calcium | cardiomyopathy | troponin

If supplemental minerals are needed to promote optimal animal performance, what is the best way of providing them: free choice or in the diet? We hypothesized that herbivores discriminate among feeds containing Na, P, and Ca and modify their choices as a function of need. One group of lambs was fed a basal diet low in P and high in Ca (low P-high Ca), whereas another group was fed a basal diet high in P and low in Ca (high P-low Ca). After 73 d of exposure to the unbalanced diets, the lambs were conditioned by offering flavored grape pomace containing NaC1, CaC[O.sub.3], or Na[H.sub.2]P[O.sub.4]. Preference for pomace + minerals was determined when all lambs were fed a basal diet of alfalfa pellets and barley grain (initial preference) and during 4 phases. Phases 1 and 2 occurred after 40 and 67 d of feeding the unbalanced basal diets, phase 3 occurred after conditioning with NaC1, CaC[O.sub.3], or Na[H.sub.2]P[O.sub.4], and phase 4 occurred 22 d after the groups were moved to 2 new (separate) locations so the animals in the different groups could not eat dirt, urine, or feces from the other pen. Preference for pomace did not differ between the groups during the initial preference tests (P = 0.62); both groups preferred NaC1 > CaC[O.sub.3] = Na[H.sub.2]P[O.sub.4] (P < 0.001). As the study progressed, and lambs fed low P-high Ca had lower P and greater Ca concentrations in serum than lambs fed high P-low Ca (P < 0.001), the preference between groups diverged. In phase 2, lambs in high P-low Ca continued to prefer NaC1 (P < 0.001), but lambs in low P-high Ca preferred Na[H.sub.2]P[O.sub.4] (P < 0.05). After conditioning, both groups preferred NaC1 = Na[H.sub.2]P[O.sub.4] > CaC[O.sub.3] (P < 0.01 to 0.11). After the groups were moved to different locations, lambs fed low P-high Ca showed the lowest concentration (3.7 mg/dL) of inorganic P in serum for all phases (P < 0.001), and they preferred Na[H.sub.2]P[O.sub.4] > NaCl = CaC[O.sub.3] (P < 0.001). In contrast, lambs in high P-low Ca avoided Na[H.sub.2]P[O.sub.4] (P < 0.05). Lambs offered high P-low Ca showed a greater preference for CaC[O.sub.3] (P = 0.12) and NaC1 (P < 0.05) and a lower preference for Na[H.sub.2]P[O.sub.4] compared with lambs fed low P-high Ca (P < 0.001). In summary, lambs discriminated among different flavored feeds containing NaC1, CaC[O.sub.3], and Na[H.sub.2]P[O.sub.4] and displayed preferences as a function of the mineral imbalance in their basal diets. Thus, it may be possible to feed Ca and P supplements free choice, such that individual animals within a group can manifest preferences based on their specific needs. Key words: calcium, learning, phosphorus, preference, sheep, sodium

Calcium signaling is critical for all cells. As a free ion ([Ca.sup.2+]), calcium links many physiological stimuli to their intracellular effectors by interacting with binding proteins whose occupancy determines the cellular effect of stimulation. Because binding site occupancy depends on the history of [Ca.sup.2+] concentration ([[Ca.sup.2+]]), [Ca.sup.2+] dynamics are critical. Calcium dynamics depend on the functional interplay between [Ca.sup.2+] transport and buffering systems whose activities depend nonlinearly on [[Ca.sup.2+]]. Thus, understanding [Ca.sup.2]. dynamics requires detailed information about these [Ca.sup.2+]. handling systems and their regulation in intact cells. However, effective methods for measuring and characterizing intracellular [Ca.sup.2+]. handling have not been available until recently. Using concepts relating voltage-gated ion-channel activity to membrane potential dynamics, we developed such methods to analyze [Ca.sup.2+] fluxes in intact cells. Here we describe this approach and applications to understanding depolarization-induced [Ca.sup.2+]. responses in sympathetic neurons.

Defecation in the nematode worm Caenorhabditis elegans is a highly rhythmic behavior that is regulated by a [Ca.sup.2+] wave generated in the 20 epithelial cells of the intestine, in part through activation of the inositol 1,4,5-trisphosphate receptor. Execution of the defecation motor program (DMP) can be modified by external cues such as nutrient availability or mechanical stimulation. To address the likelihood that environmental regulation of the DMP requires integrating distinct cellular and organismal processes, we have developed a method for studying coordinate [Ca.sup.2+] oscillations and defecation behavior in intact, freely behaving animals. We tested this technique by examining how mutations in genes known to alter [Ca.sup.2+] handling [including egl-8/phospholipase C (PLC)-[beta], kqt-3/KCNQ1, sca-1/sarco (endo)plasmic reticulum [Ca.sup.2+] ATPase, and unc-43/[Ca.sup.2+]-CaMKII] contribute to shaping the [Ca.sup.2+] wave and asked how [Ca.sup.2+] wave dynamics in the mutant backgrounds altered execution of the DMP. Notably, we find that [Ca.sup.2+] waves in the absence of PLC[beta] initiate ectopically, often traveling in reverse, and fail to trigger a complete DMP. These results suggest that the normal supremacy of the posterior intestinal cells is not obligatory for [Ca.sup.2+] wave occurrence but instead helps to coordinate the DMP. Furthermore, we present evidence suggesting that an underlying pacemaker appears to oscillate at a faster frequency than the defecation cycle and that arrhythmia may result from uncoupling the pacemaker from the DMP rather than from disrupting the pacemaker itself. We also show that chronic elevations in [Ca.sup.2+] have limited influence on the defecation period but instead alter the interval between successive steps of the DMP. Finally, our results demonstrate that it is possible to assess [Ca.sup.2+] dynamics and muscular contractions in a completely unrestrained model organism. calcium; oscillation; Caenorhabditis elegans; biosensor

Cardiac P2X purinergic receptors can mediate an increase in myocyte contractility and a potentially important role in the heart. The P2[X.sub.4] receptor (P2[X.sub.4]R) is an important subunit of native cardiac P2X receptors. With transgenic mice with cardiac-specific overexpression of P2[X.sub.4]R (Tg) used as a model, the objectives here were to characterize the P2X receptor-mediated cellular contractile and [Ca.sup.2+] transient effects and to determine the mechanism underlying the receptor-induced increase in myocyte contractility. In response to the agonist 2-methylthioATP (2-meSATP), Tg myocytes showed an increased intracellular [Ca.sup.2+] transient, as defined by fura 2 fluorescence ratio, and an enhanced contraction shortening that were unaccompanied by cAMP accumulation or L-type [Ca.sup.2+] channel activation. The increased [Ca.sup.2+] transient was not associated with any alteration in action potential duration, resting membrane potential, or diastolic fluorescence ratio or rates of rise and decline of the [Ca.sup.2+] transient. Simultaneous [Ca.sup.2+] transient and contraction measurements did not show any agonist-mediated change in myofilament [Ca.sup.2+] sensitivity. However, activation of the overexpressed P2[X.sub.4] receptor caused an enhanced SR [Ca.sup.2+] loading, as evidenced by a 2-meSATP-evoked increase in the caffeine-induced inward current and [Ca.sup.2+] transient. Similar data were obtained in wild-type mouse ventricular myocytes. Thus an increased SR [Ca.sup.2+] content, occurring in the absence of cAMP accumulation or L-type [Ca.sup.2+] channel activation, is the principal mechanism by which cardiac P2X receptor mediates a stimulatory effect on cardiac myocyte contractility. purines; calcium; sarcoplasmic reticulum

Throughout the world many people use artificial sweeteners (AS) for the purpose of reducing caloric intake. The most prominently used of these molecules include saccharin, aspartame (Nutrasweet), acesulfame-K, and cyclamate. Despite the caloric advantage they provide, one key concern in their use is their aversive aftertaste that has been characterized on a sensory level as bitter and/or metallic. Recently, it has been shown that the activation of particular T2R bitter taste receptors is partially involved with the bitter aftertaste sensation of saccharin and acesulfame-K. To more fully understand the biology behind these phenomena we have addressed the question of whether AS could stimulate transient receptor potential vanilloid-1 (TRPV1) receptors, as these receptors are activated by a large range of structurally different chemicals. Moreover, TRPV1 receptors and/or their variants are found in taste receptor cells and in nerve terminals throughout the oral cavity. Hence, TRPV1 activation could be involved in the AS aftertaste or even contribute to the poorly understood metallic taste sensation. Using [Ca.sup.2+] imaging on TRPV1 receptors heterologously expressed in the human embryonic kidney (HEK) 293 cells and on dissociated primary sensory neurons, we find that in both systems, AS activate TRPV1 receptors, and, moreover, they sensitize these channels to acid and heat. We also found that TRPV1 receptors are activated by CuS[O.sub.4], ZnS[O.sub.4], and FeS[O.sub.4], three salts known to produce a metallic taste sensation. In summary, our results identify a novel group of compounds that activate TRPV1 and, consequently, provide a molecular mechanism that may account for off tastes of sweeteners and metallic tasting salts. multisensory taste; pain; calcium imaging

Mitochondria are an important source of reactive oxygen species (ROS) formed as a side product of oxidative phosphorylation. The main sites of oxidant production are complex I and complex III, where electrons flowing from reduced substrates are occasionally transferred to oxygen to form superoxide anion and derived products. These highly reactive compounds have a well-known role in pathological states and in some cellular responses. However, although their link with [Ca.sup.2+] is well studied in cell death, it has been hardly investigated in normal cytosolic calcium concentration ([[[Ca.sup.2+]].sub.i]) signals. Several [Ca.sup.2+] transport systems are modulated by oxidation. Oxidation increases the activity of inositol 1,4,5-trisphosphate and ryanodine receptors, the main channels releasing [Ca.sup.2+] from intracellular stores in response to cellular stimulation. On the other hand, mitochondria are known to control [[[Ca.sup.2+]].sub.i] signals by [Ca.sup.2+] uptake and release during cytosolic calcium mobilization, specially in mitochondria situated close to [Ca.sup.2+] release channels. Mitochondrial inhibitors modify calcium signals in numerous cell types, including oscillations evoked by physiological stimulus. Although these inhibitors reduce mitochondrial [Ca.sup.2+] uptake, they also impair ROS production in several systems. In keeping with this effect, recent reports show that antioxidants or oxidant scavengers also inhibit physiological calcium signals. Furthermore, there is evidence that mitochondria generate ROS in response to cell stimulation, an effect suppressed by mitochondrial inhibitors that simultaneously block [[[Ca.sup.2+]].sub.i] signals. Together, the data reviewed here indicate that [Ca.sup.2+]-mobilizing stimulus generates mitochondrial ROS, which, in turn, facilitate [[[Ca.sup.2+]].sub.i] signals, a new aspect in the biology of mitochondria. Finally, the potential implications for biological modeling are discussed. mitochondria; calcium

Intracellular ANG II induces biological effects in nonrenal cells, but it is not known whether it plays a physiological role in renal proximal tubule cells (PTCs). PTCs express angiotensinogen, renin, and angiotensin-converting enzyme mRNAs, suggesting the presence of high levels of intracellular ANG II. We determined if microinjection of ANG II directly in single PTCs increases intracellular calcium concentration ([[[Ca.sup.2+]].sub.i]) and, if so, elucidated the cellular mechanisms involved. Changes in [[Ca.sup.2+]]i responses were studied by fluorescence imaging using the [Ca.sup.2+] indicator fluo 3. ANG II (1 nM) was microinjected directly in the cells, whereas cell-surface angiotensin type 1 (A[T.sub.1]) receptors were blocked by losartan (10 [micro]M). When ANG II (1 nM) was added to the perfusate, there was a marked increase in [[[Ca.sup.2+]].sub.i] that was blocked by extracellular losartan. With losartan in the perfusate, intracellular microinjection of ANG II elicited a robust increase in cytoplasmic [[[Ca.sup.2+]].sub.i] that peaked at 30 s (basal: 2.2 [+ or -] 0.3 vs. ANG II: 14.9 [+ or -] 0.4 relative fluorescence units; P < 0.01). Chelation of extracellular [Ca.sup.2+] with EGTA (2 mM) did not alter microinjected ANG II-induced [[[Ca.sup.2+].sub.i] responses ([Ca.sup.2+] free + ANG II: 12.3 [+ or -] 2.6 relative fluorescence units, not significant vs. ANG II); however, pretreatment with thapsigargin to deplete intracellular [Ca.sup.2+] stores or with U-73122 to inhibit phospholipase C (1 [micro]M each) markedly attenuated microinjected ANG II-induced [[[Ca.sup.2+].sub.i] responses. Combined microinjection of ANG II and losartan abolished [[[Ca.sup.2+]].sub.i] responses, whereas a combination of ANG II and PD-123319 had no effect. These data demonstrate for the first time that direct microinjection of ANG II in single PTCs increases [[[Ca.sup.2+]].sub.i] by stimulating intracellular A[T.sub.1] receptors and releases [Ca.sup.2+] from intracellular stores, suggesting that intracellular ANG II may play a physiological role in PTC function. intracellular calcium; kidney: microinjection; proximal tubules; receptor-mediated endocytosis

Recent genetic analysis has identified a pivotal role of primary cilia in the pathogenesis of polycystic kidney disease (PKD). However, little is known regarding how cilia loss/dysfunction contributes to cyst development. In epithelial cells, changes in apical fluid flow induce cilia-mediated [Ca.sup.2+] entry via polycystin-2 (PC2), a cation channel. The Oak Ridge Polycystic Kidney (orpk) mouse contains a mutated Tg737 gene that disrupts expression of polaris, a protein required for ciliogenesis. These studies examine the effect of cilia malformation on [Ca.sup.2+] entry in orpk cilia(-) collecting duct principal cells, and in orpk cells in which wild-type Tg737 was reintroduced, orpk cilia(+). [[[Ca.sup.2+]].sub.i] was monitored in confluent cell monolayers using fluorescence microscopy. Intrinsic apical [Ca.sup.2+] entry was measured by [Mn.sup.2+] quenching and [Ca.sup.2+] depletion/readdition under flow conditions below the threshold for stimulation. We found that unstimulated apical [Ca.sup.2+] entry was markedly increased in cilia(-) cells and was sensitive to [Gd.sup.3+], an inhibitor of PC2. Electrophysiological measurements demonstrate increased abundance of an apical channel, consistent with PC2, in cilia(-) cells. Immunofluorescence studies revealed that PC2, normally expressed on and at the base of cilia in orpk cilia(+) cells, was observed throughout the apical membrane in cilia(-) cells. Furthermore, cilia(-) cells displayed elevated subapical [Ca.sup.2+] levels measured with the near-membrane [Ca.sup.2+] indicator FFP-18. We propose that cilia exert a tonic regulatory influence on apical [Ca.sup.2+] entry, and absence of cilia results in loss of spatial organization of PC2, causing unregulated [Ca.sup.2+] entry and elevations in subapical [[Ca.sup.2+]], a factor which may contribute to cyst formation. Oak Ridge Polycystic Kidney; autosomal recessive polycystic kidney disease; polycystin-2; [Ca.sup.2+] permeability

TRPV4, a renally expressed nonselective cation channel of the transient receptor potential (TRP) family, is gated by hypotonicity. Kinases of the WNK family influence expression and function of the thiazide-sensitive [Na.sup.+]-[Cl.sup.-] cotransporter, and monogenic human hypertension has been linked to mutations in the gene coding for WNK4. Along with TRPV4, WNK isoforms are highly expressed in the distal nephron. We show here that coexpression of WNK4 downregulates TRPV4 function in human embryonic kidney (HEK-293) cells and that this effect is mediated via decreased cell surface expression of TRPV4; total abundance of TRPV4 in whole cell lysates is unaffected. The effect of the related kinase WNK1 on TRPV4 function and surface expression was similar to that of WNK4. Disease-causing point mutations in WNK4 abrogate, but do not eliminate, the inhibitory effect on TRPV4 function. In contrast to wild-type WNK4, a kinase-dead WNK4 point mutant failed to influence TRPV4 trafficking; however, deletion of the entire WNK4 kinase domain did not blunt the effect of WNK4 on localization of TRPV4. Deletion of the extreme COOH-terminal putative coiled-coil domain of WNK4 abolished its effect. In immunoprecipitation experiments, we were unable to detect direct interaction between TRPV4 and either WNK kinase. In aggregate, these data indicate that TRPV4 is functionally regulated by WNK family kinases at the level of cell surface expression. Because TRPV4 and WNK kinases are coexpressed in the distal nephron in vivo and because there is a tendency toward hypercalcemia in [TRPV4.sup.-/-] mice, we speculate that this pathway may impact systemic [Ca.sup.2+] balance. In addition, because WNK kinases and TRPV4 are activated by anisotonicity, they may comprise elements of an osmosensing or osmotically responsive signal transduction cascade in the distal nephron. hypertension; calcium balance; hypotonicity; osmoregulation

The hormone leptin and the gut peptide CCK synergistically interact to enhance the process of satiation. Although this interaction may occur at several levels of the neuroaxis, our previous results indicate that leptin can specifically enhance the satiation effect of CCK by acting on subdiaphragmatic vagal afferent neurons. Because of this localized action, we hypothesized that a high proportion of vagal afferent neurons innervating the stomach or duodenum would be responsive to leptin and/or CCK. To test this hypothesis, we measured changes in cytosolic calcium levels induced by leptin and CCK in cultured nodose ganglion neurons labeled with a retrograde neuronal tracer injected into either the stomach or the duodenum. In the neurons labeled from the stomach, CCK activated 74% (39 of 53) compared with only 35% (34 of 97) of nonlableled cells. Of the CCK-responsive neurons 60% (18 of 30) were capsaicin-sensitive. Leptin activated 42% (22 of 53) of the stomach innervating neurons compared with 26% of nonlabeled neurons. All of the leptin-sensitive neurons labeled from the stomach also responded to CCK. In the neurons labeled from the duodenum, CCK activated 71% (20 of 28). Of these CCK-responsive neurons 80% (12 of 15) were capsaicin sensitive. Leptin activated 46% (13 of 28) of these duodenal innervating neurons, of which 89% (8 of 9) were capsaicin-sensitive. Among neurons labeled from the duodenum 43% (12 of 28) were responsive to both leptin and CCK, compared with only 15% (15 of 97) of unlabeled neurons. Our results support the hypothesis that vagal afferent sensitivity to CCK and leptin is concentrated in neurons that innervate the stomach and duodenum. These specific visceral afferent populations are likely to comprise a substrate through which acute leptin/CCK interactions enhance satiation. nodose; calcium imaging; satiation; cholecystokinin

We previously showed that ablation of caveolin-1 (Cav-1) gene expression in mice promotes neointimal hyperplasia in vivo, a phenomenon normally characterized by smooth muscle cell (SMC) migration and proliferation. Whether these defects are cell autonomous, i.e., due to loss of Cav-1 within SMCs or loss of Cav-1 expression in other adjacent cell types in vivo, remains unknown. Cav-1 has been shown to associate with receptors for many vasoactive factors on the SMC surface. Therefore, Cav-1 might be an important regulator of SMC proliferation, migration, and signal transduction. To mechanistically dissect the role of Cav-1 in SMC signaling, we isolated SMCs from the aortas (AoSMCs) of Cav-1-deficient ([Cav-1.sup.-/-]) mice and characterized these cells with respect to their proliferation, migration, and [Ca.sup.2+] response to an important vasoactive factor, endothelin-1 (ET-1). 5-Bromo-2'-deoxyuridine incorporation and a wound-healing assay showed an increase in proliferation and migration rates in [Cav-1.sup.-/-] compared with wild-type ([Cav-1.sup.-/-]) AoSMCs. AoSMCs demonstrated upregulation of phosphorylated ERK1/2, cyclin D1, and proliferating cell nuclear antigen and reduced expression of the cyclin-dependent kinase inhibitor [p27.sup.Kip1]. The [Ca.sup.2+] response was examined in the presence of ET-1 and assessed by confocal microscopy with the [Ca.sup.2+]-sensitive fluorescent probe fluo 3. When treated with ET-1, Cav-[1.sup.-/-] AoSMCs exhibited a faster and larger increase in free intracellular [Ca.sup.2+] than [Cav-1.sup.+/+] cells. The ET-1-induced response in [Cav-1.sup.-/-] cells was mediated by the ETB receptor, as shown using the ETB receptor antagonist BQ-788 and the ETA receptor antagonist BQ-123. In [Cav-1.sup.-/-] cells, ETA receptor expression was reduced and E[T.sub.B] receptor expression was upregulated. Therefore, Cav-1 ablation increased the ET-1-induced [Ca.sup.2+] response in SMCs by altering the type and expression level of the ET receptor (i.e., receptor isoform switching). These data suggest a novel regulatory role for Cav-1 in SMCs with respect to their proliferation, migration, and [Ca.sup.2+]-mediated signaling. vascular disease; calcium response; endothelin receptors; neointimal hyperplasia; caveolae; caveolin

Transient receptor potential melastatin- (TRPM) and vanilloid-related (TRPV) channels are nonselective cation channels pertinent to diverse physiological functions. Multiple TRPM and TRPV channel subtypes have been identified and cloned in different tissues. However, their information in vascular tissue is scant. In this study, we sought to identify TRPM and TRPV channel subtypes expressed in rat deendothelialized intralobar pulmonary arteries (PAs) and aorta. With RT-PCR, mRNA of TRPM2, TRPM3, TRPM4, TRPM7, and TRPM8 of TRPM family and TRPV1, TRPV2, TRPV3, and TRPV4 of TRPV family were detected in both PAs and aorta. Quantitative real-time RT-PCR showed that TRPM8 and TRPV4 were the most abundantly expressed TRPM and TRPV subtypes, respectively. Moreover, Western blot analysis verified expression of TRPM2, TRPM8, TRPV1, and TRPV4 proteins in both types of vascular tissue. To examine the functional activities of these channels, we monitored intracellular [Ca.sup.2+] transients ([[[Ca.sup.2+]].sub.i]) in pulmonary arterial smooth muscle cells (PASMCs) and aortic smooth muscle cells (ASMCs). The TRPM8 agonist menthol (300 [micro]M) and the TRPV4 agonist 4[alpha]-phorbol 12,13-didecanoate (1 [micro]M) evoked significant increases in [[[Ca.sup.2+]].sub.i] in PASMCs and ASMCs. These [Ca.sup.2+] responses were abolished in the absence of extracellular [Ca.sup.2+] or the presence of 300 [micro]M [Ni.sup.2+] but were unaffected by 1 [micro]M nifedipine, suggesting [Ca.sup.2+] influx via nonselective cation channels. Hence, for the first time, our results indicate that multiple functional TRPM and TRPV channels are coexpressed in rat intralobar PAs and aorta. These novel [Ca.sup.2+] entry pathways may play important roles in the regulation of pulmonary and systemic circulation. transient receptor potential channels; calcium signaling; nonselective cation channels

1. Introduction Currently, there is a high level of commercial interest for subtilisins that work under extreme biochemical conditions [1, 2]. Therefore, understanding the structure and function of subtilisins is [...], An aprE mutant from B. subtilis 168 lacking the connecting loop [Leu.sub.75]-[Leu.sub.82] which is predicted to encode a [Ca.sup.2+] binding site was constructed. Expression of the mutant gene (aprEΔ[Leu.sub.75]-[Leu.sub.82])produced B. subtilis colonies lacking protease activity. Intrinsic fluorescence analysis revealed spectral differences between wild-type AprE and AprEΔ[L.sub.75]-[L.sub.82]. An AprEΔ[L.sub.75]-[L.sub.82] variant with reestablished enzyme activity was selected by directed evolution. The novel mutations [Thr.sub.66]Met/[Gly.sub.102]Asp located in positions which are predicted to be important for catalytic activity were identified in this variant. Although these mutations restored hydrolysis, they had no effect with respect to thermal inactivation of AprEΔ[L.sub.75]-[L.sub.82] [T.sub.66]M [G.sub.102]D. These results support the proposal that in addition to function as a calcium binding site, the loop that connects β-sheet e3 with α- helix c plays a structural role on enzyme activity of AprE from B. subtilis 168. Copyright [C] 2009 Eliel R. Romero-Garcia et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The class I myosin My01c is a mediator of adaptation of mechanoelectrical transduction in the stereocilia of the inner ear. Adaptation, which is strongly affected by [Ca.sup.2+], permits hair cells under prolonged stimuli to remain sensitive to new stimuli. Using a My01c fragment (motor domain and one IQ domain with associated calmodulin), with biochemical and kinetic properties similar to those of the native molecule, we have performed a thorough analysis of the biochemical cross-bridge cycle. We show that, although the steady-state ATPase activity shows little calcium sensitivity, individual molecular events of the cross-bridge cycle are calcium-sensitive. Of significance is a 7-fold inhibition of the ATP hydrolysis step and a 10-fold acceleration of ADP release in calcium. These changes result in an acceleration of detachment of the cross-bridge and a lengthening of the lifetime of the detached M-ATP state. These data support a model in which slipping adaptation, which reduces tip-link tension and allows the transduction channels to close after an excitatory stimulus, is mediated by My01c and modulated by the calcium transient. molecular motor | myosin

The formations of the membrane protein Ca(super 2+) -ATPase of skeletal muscle sarcoplasmic reticulum (SR) have been established for five separate conditions by X-ray crystallography. For muscular contraction, the Ca(super 2+) ions collected in muscle cells in SR are freed through Ca(super 2+) release routes, while these ions have to be pumped back into SR for muscular relaxation, a procedure controlled by Ca(super 2+) -ATPase.

The nonlinear current - voltage (I-V) characteristics are studied and the voltage-dependent tunneling conductance in nanoparticles of La2/3A1/3MnO3 (A=Ca,Sr) are analyzed. It is further shown that a distribution of grain boundary conductances is a fundamental integration, and that a small fraction of poor contacts determine the transport properties.