ATPase of basal bodies ofTetrahymena pyriformis

Summary The Ca²⁺-ATPase activity in basal bodies ofTetrahymena pyriformis was determined by cytochemical and biochemical methods. It was found that the sites of the Ca²⁺-ATPase activity are associated with the basal body microtubules. A method for the isolation of the basal bodies in a purified form in amounts for biochemical assay has been developed. Some properties of basal body ATPase were examined. It was established that the enzyme activity is specific for the hydrolysis of ATP and deoxy-ATP. The characteristic behaviour of the ATPase activity in response to divalent cations was assessed: the enzyme is stimulated by Ca²⁺ and inhibited by Mg²⁺. Basal body ATPase has a pH optimum of 7.5. Electrophoretic analysis of the protein composition of basal bodies revealed proteins with molecular weights of 56 and 43 kDa, corresponding to those of tubulin and actin respectively. It is suggested that the basal body ATPase may be involved in beating of the cilia, participating in the mechanochemical processes needed for motility ofTetrahymena pyriformis.     

Ultracytochemical localization of Ca2+-ATPase activity in the endolymphatic sac of the chick

The localization of Ca²⁺-ATPase activity was examined ultracytochemically in the endolymphatic sac (ES) of the 1-day-old chick. The reaction products showing Ca²⁺-ATPase activity were localized along the microvilli and the apical cell membranes of the epithelial cells of the ES. The lateral cell membranes also showed mild activity. These reactions were completely abolished when either Ca²⁺ or ATP was omitted from the incubation medium. The results suggest that Ca²⁺-ATPase plays a significant role as a Ca²⁺ pump for regulation of the Ca²⁺ concentration in the endolymph of the ES.     

AlF 4 − induces Ca2+ oscillations in guinea-pig ileal smooth muscle

The effects of different compounds that inhibit the isolated plasma-membrane Ca²⁺/Mg²⁺-ATPase on the cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) and on the corresponding force development have been examined in smooth muscle of the longitudinal layer of the guinea-pig ileum. F^–, in the presence of Al³⁺, induced an increase of the resting force and of the amplitude of the superimposed phasic contractions. The increase of resting force was associated with an increased level of basal [Ca²⁺]_i while the phasic contractions were accompanied by concomitant oscillations in [Ca²⁺]_i. Comparable contractions could be induced by vanadate and the calmodulin antagonist calmidazolium. The oscillations of [Ca²⁺]_i and of force elicited by AlF ₄ ^– were not modified by adrenergic or cholinergic blocking agents but were inhibited by verapamil. These phasic contractions were not affected by depleting the intracellular Ca²⁺ stores with ryanodine. This finding excludes a cytosolic origin of these oscillations. However, hyperpolarization and complete depolarization of the cells inhibited the oscillations. It is concluded that AlF ₄ ^– , vanadate and calmidazolium induce cytoplasmic Ca²⁺ oscillations possibly by acting at the plasma membrane. Indeed all these substances affect by different mechanisms the isolated plasma-membrane Ca²⁺/Mg²⁺-ATPase. The generation of membrane-linked Ca²⁺ oscillations could therefore be related to an inhibition of the plasma-membrane Ca²⁺ pump resulting in an increase of [Ca²⁺]_i. This change in [Ca²⁺]_i could be responsible for the pronounced changes of the electrical and mechanical activity of this tissue.     

Influence of caffeine,Ca2+, and Mg2+ on ryanodine depression of the tension transient in skinned myocardial fibers of the rabbit

Ryanodine, a blocker for Ca²⁺-release channels of the sarcoplasmic reticulum (SR Ca²⁺-release channels), induces depression of myocardial contraction in isolated intact muscle, which is consistent with depression of the caffeine-induced tension transient in skinned muscle fibers. In isolated SR, ryanodine binds to a specific receptor with high affinity, and this binding is enhanced by caffeine and increasing Ca²⁺ and decreased by increasing Mg²⁺. The aim of this study was to test the hypothesis that depression of myocardial contraction is mediated by changes in ryanodine-receptor binding properties. Accordingly, factors (caffeine, Ca²⁺, and Mg²⁺) affecting ryanodine-receptor binding properties in the isolated SR membrane were studied in skinned myocardial fibers from adult rabbits. The depression of the caffeine-induced tension transient by ryanodine (ryanodine depression) influenced by these three factors was measured. In a dose-dependent manner, increasing caffeine or Ca²⁺ concentrations enhanced the ryanodine depression. The concentrations for 50% ryanodine depression (IC₅₀) approximated 7mM for caffeine, and pCa 5.25 for Ca²⁺. When 1 M ryanodine and 25 mM caffeine were combined, ryanodine depression was independent of Ca²⁺ at low Ca²⁺ concentrations (20%–30% at pCa>8 and 7.5) and was a direct function of Ca²⁺ at higher concentrations (pCa 7.5–6.0 with IC₅₀ approx. pCa 6.75). In contrast, increasing Mg²⁺ reduced the ryanodine depression with IC₅₀ approximately equal to pMg 3.3. In conclusion, the caffeineor Ca²⁺-enhanced, and Mg²⁺-reduced ryanodine depression observed in this study is consistent with known ryanodinereceptor binding properties.     

Divalent cations selectively alter the voltage dependence of inactivation of A-currents in chick autonomic neurons

A-currents were studied in acutely dissociated chick autonomic neurons. Switching from salines containing 4 mM Mg²⁺ to salines containing 4 mM Ca²⁺ caused a large positive shift in the voltage dependence of steady-state inactivation, but had no effect on the voltage dependence or kinetics of activation, deactivation, the rate at which channels became inactivated, or the rate at which channels recovered from inactivation. This effect saturated with increasing concentrations of Ca²⁺. Other group IIA divalent cations were less effective than Ca²⁺, in the order Ca²⁺ >Ba²⁺ >Mg²⁺=Sr²⁺. Application of 4 mM Mg²⁺ partially antagonized the effects of 4 mM Ca²⁺. The results suggest that divalent cations selectively alter the voltage dependence of steady-state inactivation by acting at sites accessible from the outside of the cell.     

Protein kinase CK2 and regulation of Ca2+-ATPase activity in brain neuron chromatin in rats during aging

Protein kinase CK2 from neuronal chromatin phosphorylates myosin-like proteins isolated from rat brain chromatin. The protein kinase CK2 activator 1-ethyl-4,5-di(N-methylcarbamoyl)imidazole in vitro stimulated phosphorylation of myosin-like proteins and increased myosin-type Ca²⁺-ATPase activity in neuronal chromatin. After systemic administration this agent normalized Ca²⁺-ATPase activity of brain chromatin in aged rats.     

Distribution of adenosine triphosphatase activity in the developing brain of the chick embryo

The development and distribution of ⁺-K⁺-stimulated and Mg²⁺-dependent ATPase activity in different parts of the chick embryo brain were investigated. Maximal changes in the Na⁺-K⁺-stimulated ATPase activity in the hemispheres and optic lobes took place between day 15 and 17 and in the cerebellum and brain stem between day 17 and 19 of incubation. Most of this activity was localized in the membranous fraction of the chick embryo brain. An attempt was made to correlate the rapid rise in the activity of this enzyme with the development of electrical properties of the chick embryo brain.     

Effect of cytosolic Mg2+ on mitochondrial Ca2+ signaling

Cytosolic Ca²⁺ signals are followed by mitochondrial Ca²⁺ uptake, which, in turn, modifies several biological processes. Mg²⁺ is known to inhibit Ca²⁺ uptake by isolated mitochondria, but its significance in intact cells has not been elucidated. In HEK293T cells, activation of purinergic receptors with extracellular ATP caused cytosolic Ca²⁺ signals associated with parallel changes in cytosolic [Mg²⁺]. Neither signals were affected by omitting bivalent cations from the extracellular medium. The effect of store-operated Ca²⁺ influx on cytosolic Mg²⁺ concentration ([Mg²⁺]_c) was negligible. Uncaged Ca²⁺ displaced Mg²⁺ from cytosolic binding sites, but for an equivalent Ca²⁺ signal, the change in [Mg²⁺] was significantly smaller than that measured after adding extracellular ATP. Inositol 1,4,5-trisphosphate mobilized Ca²⁺ and Mg²⁺ from internal stores in permeabilized cells. The increase of [Mg²⁺] in the range that occurred in ATP-stimulated cells inhibited mitochondrial Ca²⁺ uptake in permeabilized cells without affecting mitochondrial Ca²⁺ efflux. Therefore, the Mg²⁺ signal generated by Ca²⁺ mobilizing agonists may attenuate mitochondrial Ca²⁺ uptake.     

Expression of plasma membrane Ca2+-ATPase on hepatic sinusoidal endothelial fenestrae: modification of the one-step method

The intracytoplasmic free calcium ion (Ca²⁺) concentration is maintained at a low level in mammalian tissues by extruding Ca²⁺ against a high extracellular Ca²⁺ concentration, mainly through the activity of the plasma membrane Ca²⁺-ATPase pump. The objective of the present study was to localize the plasma membrane Ca²⁺-ATPase activity on hepatic sinusoidal endothelial cells (SECs) by electron microscopic cytochemistry. The ultrastructural localization of Ca²⁺-ATPase activity on ultrathin sections of liver tissue and cultured SEC monolayer was examined by the electron microscopic cytochemical method of Ando (method A: original method) and by our modified method (method B: shortened fixation method). By method A, scanty cytochemical reaction products of Ca²⁺-ATPase were found in the SECs. By method B, Ca²⁺-ATPase activity was clearly localized on the outer surface of the plasma membrane of sinusoidal endothelial fenestrae (SEF). Our modification of Ando's method by shortening the incubation time of liver tissue or isolated SEC sections in the substrate allowed clear demonstration of Ca²⁺-ATPase activity on the SEF membrane. Use of tangential sections of primary cultures of SEC provided excellent localization results. The cytochemically reactive Ca²⁺-ATPase expressed on the SEF plasma membrane may be involved in regulation of the intracytoplasmic Ca²⁺ concentration.     

Effect of neonatal hypothyroidism on Ca-ATPase activity in the rat brain

Effect of neonatal hypothyroidism on kinetic properties of Ca²⁺-ATPase from rat brain synaptic plasma membranes and microsomes were examined. Neonatal hypothyroidism resulted in significant decrease in the enzyme activity in both the membrane systems. The synaptic membranes in control group displayed presence of one kinetic component whereas a low affinity component became evident in the hypothyroid group. In the microsomes, both control as well as hypothyroid groups showed presence of two kinetic components with the latter group showing two-fold increase in the K_m. The Ca²⁺ binding characteristics were generally unaltered in the enzyme from both the membrane systems. Our results suggest that impairment in the Ca²⁺-ATPase activity together with altered kinetic properties could be one of the underlying biochemical mechanisms leading to CNS dysfunction as a consequence of thyroid hormone deprivation during critical stages of brain development.     

Hormonal stimulation of Ca2+ and Mg2+ transport in the cortical thick ascending limb of Henle's loop of the mouse: evidence for a change in the paracellular pathway permeability

Recent studies from our laboratory have shown that in the cortical thick ascending limb of Henle's loop of the mouse (cTAL) Ca²⁺ and Mg²⁺ are reabsorbed passively, via the paracellular shunt pathway. In the present study, cellular mechanisms responsible for the hormone-stimulated Ca²⁺ and Mg²⁺ transport were investigated. Transepithelial voltages (PD_te) and transepithelial ion net fluxes (J _Na, J _Cl, J _K, J _Ca, J _Mg) were measured in isolated perfused mouse cTAL segments. Whether parathyroid hormone (PTH) is able to stimulate Ca²⁺ and Mg²⁺ reabsorption when active NaCl reabsorption, and thus PD_te, is abolished by luminal furosemide was first tested. With symmetrical lumen and bath Ringer's solutions, no Ca²⁺ and Mg²⁺ net transport was detectable, either in the absence or in the presence of PTH. In the presence of luminal furosemide and a chemically imposed lumen-to-bath directed NaCl gradient, which generates a lumen-negative PD_te, PTH slightly but significantly increased Ca²⁺ and Mg²⁺ net secretion. In the presence of luminal furosemide and a chemically imposed bath-to-lumen-directed NaCl gradient, which generates a lumen-positive PD_te, PTH slightly but significantly increased Ca²⁺ and Mg²⁺ net reabsorption. In view of the observed small effect of PTH on passive Ca²⁺ and Mg²⁺ movement, a possible interference of furosemide with the hormonal response was considered. To investigate this possibility, Ca²⁺ and Mg²⁺ transport was first stimulated with PTH in tubules under control conditions. Then active NaCl reabsorption was abolished by furosemide and the effect of PTH on J _Ca and J _Mg measured. In the absence of PD_te and under symmetrical conditions, no Ca²⁺ and Mg²⁺ transport was detectable, either in the presence or absence of PTH. In the presence of a bath-to-lumen-directed NaCl gradient, Ca²⁺ and Mg²⁺ reabsorption was significantly higher in the presence than in the absence of PTH. Finally, when active NaCl transport was not inhibited by furosemide, but reduced by a bath-to-lumen-directed NaCl gradient, PTH strongly increased J _Ca and J _Mg, whereas only a small increase in PD_te was noted. In conclusion, these data suggest that PTH exerts a dual action on Ca²⁺ and Mg²⁺ transport in the mouse cTAL by increasing the transepithelial driving force for Ca²⁺ and Mg²⁺ reabsorption through hormone-mediated PD_te alterations and by modifying the passive permeability for Ca²⁺ and Mg²⁺ of the epithelium, very probably at the level of the paracellular shunt pathway.     

New insights into the dynamics of sinusoidal endothelial fenestrae in liver sinusoidal endothelial cells

Ultrastructural studies have shown that liver sinusoidal endothelial cells (LSECs) contain a cytoskeletal framework of filamentous actin, and that the presence of actin in the form of a calmodulin—actomyosin complex is responsible for regulation of the diameter of sinusoidal endothelial fenestrae (SEF). Rho has emerged as an important regulator of the actin cytoskeleton and consequently of cell morphology. We investigated actin filaments in relation to SEF in LSEC using heavy meromyosin decorated reaction and elucidated the roles of Rho and actin cytoskeleton in morphological and functional alterations of SEF. Second, according to intracytoplasmic Ca²⁺ concentration, plasma membrane Ca²⁺Mg²⁺-ATPase activities were clearly demonstrated on the outer surface of the labyrinth-like SEF in the isolated LSECs. Furthermore, by investigating intracytoplasmic Ca²⁺ concentration, we have demonstrated plasma membrane Ca²⁺-Mg²⁺-ATPase activities on the outer surface of the labyrinth-like SEF in the isolated LSECs. Currently, the majority of fenestral studies are focused on finding ways to increase the liver sieve’s porosity, which is reduced through pathological mechanisms. Dr. Hiroaki Yokomori, of the Department of Internal Medicine, Kitasato Medical Center Hospital, Saitama, Japan, is the winner of the Japanese Society for Clinical Molecular Morphology Award for Promoting Young Researchers in 2007. Dr. Yokomori was recognized for his great contribution in elucidating the role of sinusoidal endothelial fenestrae in the physiology and pathology of the liver.     

Discrimination of Ca2+-ATPase activity of the sarcoplasmic reticulum from actomyosin-type ATPase activity of myofibrils in skinned mammalian skeletal muscle fibres: distinct effects of cyclopiazonic acid on the two ATPase activities

Summary We have developed a procedure to discriminate actomyosin-type ATPase activity from Ca²⁺-ATPase activity of sarcoplasmic reticulum (SR) in mechanically skinned fibres, determining simultaneously their Ca²⁺-induced tension and accompanying ATPase activity. When they were treated with an alkaline CyDTA-containing solution of low ionic strength which was reported to remove troponin C, the fibres showed a considerable amount of Ca²⁺-dependent ATPase activity, in spite of having little or no Ca²⁺-induced isometric tension. The residual ATPase activity is ascribed to the Ca²⁺-ATPase activity of SR, because it is completely abolished by 1% CHAPS treatment for 10 min. This conclusion is also supported by the finding that the Ca²⁺-dependence of the ATPase activity is very similar to that of Ca²⁺-ATPase of SR isolated from rabbit skeletal muscle, and that the estimated activity is consistent with the reported values of direct determinations. On the other hand, treatment with a detergent such as CHAPS or Triton X-100 removes SR activities (ATPase and Ca-uptake), leaving Ca²⁺-induced tension and actomyosin-type ATPase activity unchanged. This procedure indicated that the contribution of Ca²⁺-ATPase activity of SR may be minimal in total steady-state ATPase activity of mechanically skinned mammalian skeletal muscle fibres. Successive CyDTA and CHAPS treatments eliminated both Ca²⁺-induced tension and ATPase activity, which were recovered by the addition of troponin C. Using these procedures, we also examined the effect of cyclopiazonic acid (CPA) which was reported to be a specific inhibitor of Ca²⁺-ATPase of SR. Ca²⁺-ATPase activity of SR in skinned fibres was inhibited completely by 10 m CPA and held to one-half by about 0.2 m. This effect was only partially reversible. CPA at 10 m or higher concentrations showed Ca²⁺-sensitizing action on myofibrils, which was readily reversible. CPA at 3 m inhibited almost completely the Ca²⁺-ATPase activity of SR, while it had no effect on either actomyosin-type ATPase or isometric tension of myofibrils.     

The role of sarcoplasmic reticulum and sarcoplasmic reticulum Ca2+-ATPase in the smooth muscle tone of the cat gastric fundus

Circular smooth muscle strips isolated from cat gastric fundus were studied in order to understand whether the sarcoplasmic reticulum (SR) and SR Ca²⁺-ATPase could play a role in the regulation of the muscle tone. Cyclopiazonic acid (CPA), a specific inhibitor of SR Ca²⁺-ATPase, caused a significant and sustained increase in muscle tone, depending on the presence of extracellular Ca²⁺. Nifedipine and cinnarizin only partially suppressed the CPA-induced tonic contraction. Bay K 8644 antagonized the relaxant effect of nifedipine in CPA-contracted fundus. Nitric-oxide-releasing agents sodium nitroprusside and 3-morpholino-sydnonimine completely suppressed the CPA-induced tonic contraction. The blockers of Ca²⁺-activated K⁺ channels, tetraethylammonium, charybdotoxin and/or apamin, decreased the contractile effect of CPA. Vanadate increased the tone but did not change significantly the effect of CPA. CPA exerted its contractile effect even when Ca²⁺ influx was triggered through the Na⁺/Ca²⁺ exchanger and the other Ca²⁺ entry pathways were blocked. Thapsigargin, another specific SR Ca²⁺-ATPase inhibitor, also increased the muscle tone. The effect of thapsigargin was completely suppressed by sodium nitroprusside and 3-morpholino-sydnonimine and partially by nifedipine. In conclusion, under conditions when the SR Ca²⁺-ATPase is inhibited, the tissue develops a strong tonic contraction and a large part of this is mediated by Ca²⁺ influx presumably via nifedipine-sensitive Ca²⁺ channels. This study suggests the important role of SR Ca²⁺-ATPase in the modulation of the muscle tone and the function of SR as a “buffer barrier” to Ca²⁺ entry in the cat gastric fundus smooth muscle. Received: 10 August 1995/Received after revision: 9 November 1995/Accepted: 10 November 1995     

Calcium binding and fluorescence measurements of dansylaziridine-labelled troponin C in reconstituted thin filaments

Summary The direct binding of Ca²⁺ to reconstituted thin filaments containing troponin C and the 5-dimethylaminonaphthalene-1-sulphonylaziridine (DANZ) fluorescent analogue of troponin C (TnC _DANZ ) was measured (25° C) at three Mg²⁺ concentrations. Biphasic Scatchard plots were found for all binding curves reflecting the binding of Ca²⁺ to high- and low-affinity sites of troponin. The binding of Ca²⁺ to the high-affinity sites had a greater sensitivity to Mg²⁺ (K _Mg=1×10⁴ m ^–1) than the low-affinity sites (K _Mg=1.2×10³ m ^–1). The fluorescence change of thin filaments reconstituted with TnC _DANZ was titrated with Ca²⁺ in the same solutions used for binding assays. The Ca²⁺-dependent fluorescence change had nearly the same sensitivity to Mg²⁺ (K _Mg=9.4×10² m ^–1) as did Ca²⁺ binding to the low-affinity sites. The Ca²⁺ concentration at the midpoint of the fluorescence change was about 0.3 log units less than at the midpoint for Ca²⁺ binding to the low-affinity sites. A similar relationship between the fluorescence change and Ca²⁺ binding to the low-affinity sites of isolated TnC _DANZ was measured (4° C). From these results the binding of Ca²⁺ to either low-affinity site is concluded to produce the fluorescence change. In comparison with the low-affinity sites of isolated troponin and troponin-tropomyosin complex, the low-affinity sites of reconstituted thin filaments were consistently lower in Ca²⁺ affinity.     

Magnesium dependent contraction of glycerinated smooth muscle

The effect of Mg²⁺ on the contraction of glycerinated smooth and skeletal muscles was examined. In the absence of Ca²⁺, glycerinated smooth muscle contracted slowly when exposed to 5 mM adenosine triphosphate (ATP) and Mg²⁺. The magnitude of this contraction depended on the concentration of Mg²⁺ in a range of 1–20 mM. When 20 mM Mg²⁺ was used with ATP, glycerinated smooth muscle contracted almost maximally in a Ca²⁺ free environment, and it did not contract further on subsequent addition of 0.1 mM of free Ca²⁺ (EGTA-CaEGTA buffer). In contrast, in skeletal muscle a Ca²⁺ dependent contraction could always be elicited in the presence of 1–20 mM Mg²⁺ and 5 mM ATP. Tension development in smooth muscle induced by Mg²⁺ and ATP was not due to a contamination by Ca²⁺ or the removal of a Ca²⁺-sensitive protein. In the present experimental conditions, glycerinated smooth muscle developed a Mg²⁺-dependent contraction in a Ca²⁺ free medium, but glycerinated skeletal muscle did not.     

Ca entry through a non-selective cation channel in Aplysia bag cell neurons

Ca²⁺ influx through voltage-gated Ca²⁺ channels is a fundamental signaling event in neurons; however, non-traditional routes, such as non-selective cation channels, also permit Ca²⁺ entry. The present study examines the Ca²⁺ permeability of a cation channel that drives an afterdischarge in Aplysia bag cell neurons. The firing of these neurons induces peptide release and reproduction. Single channel-containing inside-out patches excised from cultured bag cell neurons, with the cytoplasmic face bathed in K⁺-aspartate and the extracellular face bathed in artificial seawater (11 mM Ca²⁺), had a reversal potential near +50 mV. In keeping with Ca²⁺ permeability, this was right-shifted to approximately +60 mV in high Ca²⁺ (substituted for Mg²⁺) and left-shifted to around +40 mV in zero Ca²⁺ (replaced with Mg²⁺). The current showed inward rectification between +30 and +90 mV, and a conductance of 29 pS in normal Ca²⁺, 30 pS in high Ca²⁺, 32 pS in Ba²⁺ (substituted for Ca²⁺), but only 21 pS in zero Ca²⁺. Despite a greater conductance in Ba²⁺, the channel did not display anomalous mol fraction in an equimolar Ca²⁺–Ba²⁺ mix. Eliminating internal Mg²⁺ lowered activity, but did not alter inward rectification, suggesting intracellular Mg²⁺ is a fast, voltage-independent blocker. Imaging bag cell neurons in Mn²⁺ saline (substituted for Ca²⁺) revealed enhanced fura-quench following cation channel activation, consistent with Mn²⁺ permeating as a Ca²⁺ surrogate. Finally, triggering the cation channel while tracking capacitance revealed a Ca²⁺-dependent increase in membrane surface area, consistent with vesicle fusion. Thus, the cation channel not only drives the afterdischarge, but also passes Ca²⁺ to potentially initiate secretion. In general, this may represent an alternate means by which neurons elicit neuropeptide release.     

Enrichment and partial enzyme characterization of ATPase activity associated with the outward-facing membrane complex and inward-facing membrane of the surface epithelial syncytium of Schistosoma mansoni

The outward-facing (OFM) and inward-facing (IFM) membranes of the surface epithelial syncytium of Schistosoma mansoni were separated by sequential exposure to saponin solutions. The OFM, containing both inner and outer bilayers, contained ATPase activity that was stimulated by Mg²⁺ and Na⁺, but not K⁺ or HCO₃^?, and was inhibited by Ca²⁺ and ethacrynic acid. The OFM enzyme was unaffected by ouabain, oligomycin, SCN^? and azide and had a pH optimum of 7.5. The OFM ATPase therefore has properties similar to ATPases characterized from the apical membrane of a variety of epithelial cells where it is thought to augment the regulatory cell volume decreasing function of (Na⁺+K⁺)Mg²⁺?ATPase. The IFM contained ATPase activity that was stimulated by Mg²⁺, Na⁺ and K⁺, and was inhibited by ouabain indicating that the IFM enzyme was the Na⁺-pump ATPase. The results are discussed in terms of the transepithelial transport function of the surface epithelial syncytium and a Ca²⁺-ATPase reported previously from the OFM of S. mansoni.     

Ca2+ bound to the high affinity divalent cation-binding site of actin enhances actophorin-induced depolymerization of muscle F-actin but inhibits actophorin-induced depolymerization of Acanthamoeba F-actin

Summary The cation tightly bound to actin, Mg²⁺ or Ca²⁺, affects the ability of actophorin to accelerate depolymerization of filaments and bind to monomers of actin prepared from rabbit skeletal muscle and Acanthamoeba castellanii. Actophorin interacted similarly with muscle and Acanthamoeba Mg²⁺-F-actin but depolymerized muscle Mg²⁺-F-actin more efficiently. Muscle Ca^2+-F-actin depolymerized about 5 times more rapidly than Mg²⁺-F-actin in the presence of actophorin but Acanthamoeba Ca^2+-F-actin was highly resistant to actophorin. Muscle actin subunits dissociated more rapidly than Acanthamoeba actin subunits from copolymers of muscle and Acanthamoeba Ca²⁺-actin upon addition of actophorin although Acanthamoeba actin dissociated much more rapidly from copolymers than from its homopolymer. The Kd of the 1:1 complex between actophorin and monomeric actin was somewhat lower for muscle Mg²⁺-ATP-G-actin than for both Acanthamoeba Mg²⁺-ATP-G-actin and muscle Ca²⁺-ATP-G-actin. The data for the interactions of actophorin with Acanthamoeba Ca²⁺-ATP-G-actin or muscle and amoeba Mg²⁺-and Ca²⁺-ADP-G-actin were incompatible with the formation of 1:1 actin:actophorin complexes and, thus, Kd values could not be calculated. While it may not be surprising that actophorin would interact differently with Mg²⁺-and Ca²⁺-actin, it is unexpected that the nature of the tightly bound cation would have such dramatically opposite effects on the ability of actophorin to depolymerize muscle and Acanthamoeba F-actin. Differential severing by actophorin, with Acanthamoeba Ca²⁺-actin being almost totally resistant, is sufficient to explain the results but other possibilities cannot be ruled out.     

The mammalian melastatin-related transient receptor potential cation channels: an overview

The mammalian melastatin-related transient receptor potential (TRPM) subfamily contains eight members. TRPM proteins, consisting of six putative transmembrane domains and intracellular N and C termini, form monovalent-permeable cation channels with variable selectivity for Ca²⁺, Mg²⁺ and other divalent cations. Some functions are linked to their individual cation selectivity: the highly divalent-permeable cation channels TRPM6 and TRPM7 are involved in the control of Mg²⁺ influx, whereas the Ca²⁺-impermeable channels TRPM4 and TRPM5 modulate cellular Ca²⁺ entry by determining the membrane potential. TRPM2, TRPM3 and TRPM8 mediate a direct influx of Ca²⁺ in response to specific stimuli. Electrophysiological properties of the founding member, melastatin (TRPM1), are unexplored. The individual TRPM members are activated by different stimuli, including voltage, Ca²⁺, temperature, cell swelling, lipid compounds and other endogenous or exogenous ligands. This review summarizes molecular features, activation mechanisms, biophysical properties and modulators of TRPM channels.