Amino acid sequences of two immune-dominant epitopes of recoverin are involved in Ca<Superscript>2+</Superscript>/recoverin-dependent inhibition of phosphorylation of rhodopsin

Antibodies AB^60–72 and AB^80–92 against two immune-dominant epitopes of photoreceptor Ca²⁺-binding protein recoverin, 60-DPKAYAQHVFRSF-72 and 80-LDFKEYVIALHMT-92, which can be exposed in a Ca²⁺-dependent manner, were obtained. The presence of AB^60–72 or AB^80–92 results in a slight increase in Ca²⁺-affinity of recoverin and does not affect significantly a Ca²⁺-myristoyl switch mechanism of the protein. However in the presence of AB^60–72 or AB^80–92 recoverin loses its ability to interact with rhodopsin kinase and consequently to perform a function of Ca²⁺-sensitive inhibitor of rhodopsin phosphorylation in photoreceptor cells.     

Monoclonal antibodies to the Ca2+ + Mg2+-dependent ATPase of sarcoplasmic reticulum identify polymorphic forms of the enzyme and indicate the presence in the enzyme of a classical high-affinity Ca2+ binding site

In order to determine whether polymorphic forms of the Ca²⁺ + Mg²⁺-dependent ATPase exist, we have examined the cross-reactivity of five monoclonal antibodies prepared against the rabbit skeletal muscle sarcoplasmic reticulum enzyme with proteins from microsomal fractions isolated from a variety of muscle and nonmuscle tissues. All of the monoclonal antibodies cross-reacted in immunoblots against rat skeletal muscle Ca²⁺ + Mg²⁺-dependent ATPase but they cross-reacted differentially with the enzyme from chicken skeletal muscle. No cross-reactivity was observed with the Ca²⁺ + Mg²⁺-dependent ATPase of lobster skeletal muscle. The pattern of antibody cross-reactivity with a 100,000 dalton protein from sarcoplasmic reticulum and microsomes isolated from various muscle and nonmuscle tissues of rabbit demonstrated the presence of common epitopes in multiple polymorphic forms of the Ca²⁺ + Mg²⁺-dependent ATPase. One of the monoclonal antibodies prepared against the purified Ca²⁺ + Mg²⁺-dependent ATPase of rabbit skeletal muscle sarcoplasmic reticulum was found to cross-react with calsequestrin and with a series of other Ca²⁺-binding proteins and their proteolytic fragments. Its cross-reactivity was enhanced in the presence of EGTA and diminished in the presence of Ca²⁺. Its lack of cross-reactivity with proteins that do not bind Ca²⁺ suggests that it has specificity for antigenic determinants that make up the Ca²⁺-binding sites in several Ca²⁺-binding proteins including the Ca²⁺ + Mg²⁺-dependent ATPase.This paper is dedicated to the memory of Dr. David E. Green.     

Nicotine-induced Ca<Superscript>2+</Superscript>-myristoyl Switch of Neuronal Ca<Superscript>2+</Superscript> Sensor VILIP-1 in Hippocampal Neurons: A Possible Crosstalk Mechanism for Nicotinic Receptors

Visinin-like protein (VILIP-1) belongs to the neuronal Ca²⁺ sensor family of EF-hand Ca²⁺-binding proteins that regulate a variety of Ca²⁺-dependent signal transduction processes in neurons. It is an interaction partner of α4β2 nicotinic acetylcholine receptor (nAChR) and increases surface expression level and agonist sensitivity of the receptor in oocytes. Nicotine stimulation of nicotinic receptors has been reported to lead to an increase in intracellular Ca²⁺ concentration by Ca²⁺-permeable nAChRs, which in turn might lead to activation of VILIP-1, by a mechanism described as the Ca²⁺-myristoyl switch. It has been postulated that this will lead to co-localization of the proteins at cell membranes, where VILIP-1 can influence functional activity of α4-containing nAChRs. In order to test this hypothesis we have investigated whether a nicotine-induced and reversible Ca²⁺-myristoyl switch of VILIP-1 exists in primary hippocampal neurons and whether pharmacological agents, such as antagonist specific for distinct nAChRs, can interfere with the Ca²⁺-dependent membrane localization of VILIP-1. Here we report, that only α7- but not α4-containing nAChRs are able to elicit a Ca²⁺-dependent and reversible membrane-translocation of VILIP-1 in interneurons as revealed by employing the specific receptor antagonists dihydro-beta-erythroidine and methylallylaconitine. The nAChRs are associated with processes of synaptic plasticity in hippocampal neurons and they have been implicated in the pathology of CNS disorders, including Alzheimer’s disease and schizophrenia. VILIP-1 might provide a novel functional crosstalk between α4- and α7-containing nAChRs.     

Analysis of the Ca<Superscript>2+</Superscript> response of mycelial fungi to external effects by the recombinant aequorin method

Using the mutant strain Aspergillus awamori 66A, producing the recombinant Ca²⁺-dependent photosensitive protein aequorin, the dynamics of Ca²⁺ was studied for the first time in the cytosol of micromycetes exposed to stressful factors, such as an increase in extracellular Ca²⁺ to 50 mM, hypoosmotic shock, and mechanical shock. The cell response to stress proved to involve an increase in the Ca²⁺ concentration in the cytosol, which was determined from the amplitude of aequorin luminescence and the time of the amplitude enhancement and relaxation. The level of the Ca²⁺ response depended on the physiological stimulus. Inhibitory analysis with various agents that block Ca²⁺ channels and with agonists that specifically enhance the activity of the channels suggested that (1) the level of Ca²⁺ in the cytosol of micromycetes increases in response to stress because of the ion influx from both the growth medium and intracellular reservoirs and (2) potential-dependent transport systems play the major role in the Ca²⁺ influx into the cytosol of the micromycete cells.Translated from Mikrobiologiya, Vol. 73, No. 6, 2004, pp. 734–740.Original Russian Text Copyright © 2004 by Kozlova, Egorov, Kupriyanova-Ashina, Rid, El-Registan.     

Mitochondrial Ca<Superscript>2+</Superscript> cycle mediated by the palmitate-activated cyclosporin a-insensitive pore

Earlier we found that in isolated rat liver mitochondria the reversible opening of the mitochondrial cyclosporin A-insensitive pore induced by low concentrations of palmitic acid (Pal) plus Ca²⁺ results in the brief loss of Δψ [Mironova et al., J Bioenerg Biomembr (2004), 36:171–178]. Now we report that Pal and Ca²⁺, increased to 30 and 70 nmol/mg protein respectively, induce a stable and prolonged (10 min) partial depolarization of the mitochondrial membrane, the release of Ca²⁺ and the swelling of mitochondria. Inhibitors of the Ca²⁺ uniporter, ruthenium red and La³⁺, as well as EGTA added in 10 min after the Pal/Ca²⁺-activated pore opening, prevent the release of Ca²⁺ and repolarize the membrane to initial level. Similar effects can be observed in the absence of exogeneous Pal, upon mitochondria accumulating high [Sr²⁺], which leads to the activation of phospholipase A₂ and appearance of endogenous fatty acids. The paper proposes a new model of the mitochondrial Ca²⁺ cycle, in which Ca²⁺ uptake is mediated by the Ca²⁺ uniporter and Ca²⁺ efflux occurs via a short-living Pal/Ca²⁺-activated pore.     

Effect of a chemical analogue of autoinducers of microbial anabiosis on the Ca<Superscript>2+</Superscript> response of mycelial fungi

The microbial alkylhydroxybenzenes (AHB), which are anabiosis autoinducers also termed d₁ factors, participate in the stress response of mycelial fungi, as determined from changes in intracellular Ca²⁺ concentration. By using the genetically modified strain Aspergillus awamori 66A, which produces the recombinant Ca²⁺-dependent protein aequorin, the dynamics of Ca²⁺ was studied in the cytosol of cells exposed to mechanical shock in the presence of protective doses (0.001–0.01% w/vol) of a chemical AHB analogue, 4-n-hexylresorcinol. As under stressful conditions, Ca²⁺ concentration increases in the cell cytosol in response to an enhanced AHB level in a growing fungal culture; thus, AHB is perceived by cells as a stress signal. The level of cell response, which was determined from the amplitude of luminescence dependent on the Ca²⁺ concentration in the cytosol, was related to the physiological age of the cells and the AHB concentration. Micromycete preincubation with AHB was found to protect cells from subsequent stress; this was reflected in the Ca²⁺ response. The protective AHB effect was manifested as (1) a significant decrease in the amplitude of luminescence and, thus, in Ca²⁺ accumulation in the cytosol during subsequent mechanical stress (as compared to the control—mechanical stress only); (2) development of a secondary Ca²⁺ response, which was not observed in the control; and (3) a high level of Ca²⁺ retained in the cytosol for a long time in the presence of AHB (as compared to the control without preincubation with AHB). The mechanisms underlying the AHB effect on Ca²⁺ transport systems are discussed.Translated from Mikrobiologiya, Vol. 73, No. 6, 2004, pp. 741–750.Original Russian Text Copyright © 2004 by Kozlova, Kupriyanova-Ashina, Egorov, El-Registan.     

Point amino acid substitutions in the Ca2+-binding sites of recoverin: II. The unusual behavior of the protein upon the binding of calcium ions

The structural properties of myristoylated forms of recombinant recoverin of the wild type and of its mutants with damaged second and/or third Ca²⁺-binding sites were studied by fluorimetry and circular dichroism. The interaction of wild-type recoverin with calcium ions was shown to induce unusual structural rearrangements in its molecule. In particular, protein binding with Ca²⁺ ions results in an increase in the mobility of the environment of Trp residues, in hydrophobicity, and in thermal stability (its thermal transition shifts by 15°C to higher temperatures) but has almost no effect on its secondary structure. Similar structural changes induced by Ca²⁺ are also characteristic of the -EF2 mutant of recoverin whose second Ca²⁺-binding site is modified and cannot bind calcium ions. The structural properties of the -EF3 and -EF2,3 mutants (whose third or simultaneously second and third Ca²⁺-binding sites, respectively, are modified and damaged) are practically indifferent to the presence of calcium ions. For the communication I, see [1].     

Intracellular Mg<Superscript><Emphasis Type="Bold">2+</Emphasis></Superscript> Influences Both Open and Closed Times of a Native Ca<Superscript><Emphasis Type="Bold">2+</Emphasis></Superscript>-activated BK Channel in Cultured Human Renal Proximal Tubule Cells

Effects of intracellular Mg²⁺ on a native Ca²⁺-and voltage-sensitive large-conductance K⁺ channel in cultured human renal proximal tubule cells were examined with the patch-clamp technique in the inside-out mode. At an intracellular concentration of Ca²⁺ ([Ca²⁺]_i) of 10⁻⁵–10⁻⁴ M, addition of 1–10 mM Mg²⁺ increased the open probability (P_o) of the channel, which shifted the P_o –membrane potential (V_m) relationship to the negative voltage direction without causing an appreciable change in the gating charge (Boltzmann constant). However, the Mg²⁺-induced increase in P_o was suppressed at a relatively low [Ca²⁺]_i (10^−5.5–10⁻⁶ M). Dwell-time histograms have revealed that addition of Mg²⁺ mainly increased P_o by extending open times at 10⁻⁵ M Ca²⁺ and extending both open and closed times simultaneously at 10^−5.5 M Ca²⁺. Since our data showed that raising the [Ca²⁺]_i from 10⁻⁵ to 10⁻⁴ M increased P_o mainly by shortening the closed time, extension of the closed time at 10^−5.5 M Ca²⁺ would result from the Mg²⁺-inhibited Ca²⁺-dependent activation. At a constant V_m, adding Mg²⁺ enhanced the sigmoidicity of the P_o–[Ca²⁺]_i relationship with an increase in the Hill coefficient. These results suggest that the major action of Mg²⁺ on this channel is to elevate P_o by lengthening the open time, while extension of the closed time at a relatively low [Ca²⁺]_i results from a lowering of the sensitivity to Ca²⁺ of the channel by Mg²⁺, which causes the increase in the Hill coefficient. M. Kubokawa and Y. Sohma contributed equally to this work.     

Point amino acid substitutions in the Ca2+-binding sites of recoverin: III. A mutant with the fourth reconstructed Ca2+-binding site

Unlike wild type recoverin with only two (the second and the third) functioning Ca⁺²-binding sites out of four potential ones, the +EF4 mutant contains a third active Ca⁺²-binding site. This site was reconstructed from the fourth potential Ca⁺²-binding domain by the introduction of several amino acid substitutions in it by site-directed mutagenesis. The effect of these mutations in the fourth potential Ca⁺²-binding site of myristoylated recoverin on the structural features and conformational stability of the protein was studied by fluorimetry and circular dichroism. The apoform of the resulting mutant (free of Ca²⁺ ions) was shown to have a higher calcium capacity, significantly lower thermal stability, and noticeably different secondary and tertiary structures as compared with the apoform of wild-type recoverin. For communication II, see [1].     

Recombinant human erythropoietin induces proliferation and Ca<Superscript>2+</Superscript>-influx in specific leukocyte subpopulations of rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>) blood and head kidney cells

In mammals, erythropoietin regulates the development and differentiation of erythrocytes. Although hematopoietic cells of bony fish correspond in their ontogeneic development, morphology, and function to their mammalian counterparts, an erythropoietin (EPO)-like molecule has not been identified. In this study we present evidence for a mitogenic response of blood and head kidney leukocytes of rainbow trout after stimulation by recombinant human EPO (rhu EPO). The modulation of cellular activities is accompanied by the induction of DNA-binding activities in nuclear extracts of these cells. In addition, flow cytometric analysis of intracellular Ca²⁺ concentrations revealed a long-lasting and rhu EPO dose-dependent increase, which was shown to be abrogated by cross-aggregation of surface IgM using anti-trout-IgM monoclonal antibodies (mabs). In flow cytometric dual-labeling experiments using rhu EPO/anti-EPO antiserum and mabs specific for trout leukocyte subpopulations, it was shown that a subpopulation of trout B-cells binds rhu EPO. Moreover, in a modified Ca²⁺ activation assay, it was demonstrated that this blood B-cell subpopulation is the rhu EPO responder population. In conclusion, the data suggest the existence of EPO-binding receptors in trout that are able to trigger Ca²⁺-independent intracellular signaling in hematopoietic cells of head kidney and Ca²⁺-dependent activation of a subpopulation of B-lymphocytes.Abbreviations FSC forward scatter - IFN interferon - IL interleukin - mab monoclonal antibody - PBL peripheral blood leukocytes - PHA phythemagglutitnin - rhu EPO recombinant human erythropoietin - SSC side scatter - TNF tumor necrosis factor Communicated by G. Heldmaier     

L-type Ca<Superscript>2+</Superscript> channel and Ca<Superscript>2+</Superscript>-permeable nonselective cation channel as a Ca<Superscript>2+</Superscript> conducting pathway in myocytes isolated from the cricket lateral oviduct

The Ca²⁺-conducting pathway of myocytes isolated from the cricket lateral oviduct was investigated by means of the whole-cell patch clamp technique. In voltage-clamp configuration, two types of whole cell inward currents were identified. One was voltage-dependent, initially activated at –40 mV and reaching a maximum at 10 mV with the use of 140 mM Cs²⁺-aspartate in the patch pipette and normal saline in the bath solution. Replacement of the external Ca²⁺ with Ba²⁺ slowed the current decay. Increasing the external Ca²⁺ or Ba²⁺ concentration increased the amplitude of the inward current and the current–voltage (I–V) relationship was shifted as expected from a screening effect on negative surface charges. The inward current could be carried by Na⁺ in the absence of extracellular Ca²⁺. Current carried by Na⁺ (I _Na) was almost completely blocked by the dihydropyridine Ca²⁺ channel antagonist, nifedipine, suggesting that the I _Na is through voltage-dependent L-type Ca²⁺ channels. The other inward current is voltage-independent and its I–V relationship was linear between –100 mV to 0 mV with a slight inward rectification at more hyperpolarizing membrane potentials when 140 mM Cs⁺-aspartate and 140 mM Na⁺-gluconate were used in the patch pipette and in the bath solution, respectively. A similar current was observed even when the external Na⁺ was replaced with an equimolar amount of K⁺ or Cs⁺, or 50 mM Ca²⁺ or Ba²⁺. When the osmolarity of the bath solution was reduced by removing mannitol from the bath solution, the inward current became larger at negative potentials. The I–V relationship for the current evoked by the hypotonic solution also showed a linear relationship between –100 mV to 0 mV. Bath application of Gd³⁺ (10 M) decreased the inward current activated by membrane hyperpolarization. These results clearly indicate that the majority of current activated by a membrane hyperpolarization is through a stretch-activated Ca²⁺-permeable nonselective cation channel (NSCC). Here, for the first time, we have identified voltage-dependent L-type Ca²⁺ channel and stretch-activated Ca²⁺-permeable NSCCs from enzymatically isolated muscle cells of the cricket using the whole-cell patch clamp recording technique.Abbreviations I _Ca Ca²⁺ current - I _Na Na⁺ current - I–V current–voltage - NSCC nonselective cation channel Communicated by G. Heldmaier     

T-channels and Na<Superscript>+</Superscript>,Ca<Superscript>2+</Superscript>-exchangers as components of the Ca<Superscript>2+</Superscript>-system of regulation of activity of the heart myocardium of the frog <Emphasis Type="Italic">Rana temporaria</Emphasis>

Earlier we have shown that regulation of rhythm and strength of the frog heart contractions, mediated by transmitters of the autonomic nervous system, is of the Ca²⁺-dependent character. In the present work, we studied chronoand inotropic effect of verapamil—an inhibitor of Ca²⁺-channels of the L-type, of nickel chloride-an inhibitor of Ca²⁺—channels of the T-type and of Na⁺,Ca²⁺exchangers as well as of adrenaline and acetylcholine (ACh) after nickel chloride. It has been found that the intracardially administered NiCh₂ at a dose of 0.01 μg/kg produced a sharp fall of amplitude of action potential (AP) and an almost twofold deceleration of heart rate (HR). The intracardiac administration of NiCh₂ (0.01 μg/kg) on the background of action of verapamil (6 mg/kg, i/m) led as soon as after 3 min to even more prominent HR deceleration and to further fall of the AP amplitude by more than 50% as compared with norm. An intracardiac administration of adrenaline (0.5 mg/kg) partly restored the cardiac activity. However, preservation of the myocardium electrical activity in such animals was brief and its duration did not exceed several minutes. Administration of Ni²⁺ on the background of acetylcholine (3.6 mg/kg) led to almost complete cessation of cardiac activity. As soon as 3 min after injection of this agent the HR decreased to 2 contractions/min. On electrograms (EG), the 10-fold fall of the AP amplitude was recorded. To elucidate role of extraand intracellular Ca²⁺ in regulation of strength of heart contractions, isometric contraction of myocardium preparations was studied in response to action of NiCl₂ (10–200 μM), verapamil (70 μM), adrenaline (5 μM), and acetylcholine (0.2 μM) after NiCl₂. It has been found that Ni²⁺ causes a dose-dependent increase of the muscle contraction amplitude. Minimal change of the contraction amplitude (on average, by 14.9% as compared with control) was recorded at a Ni²⁺ concentration of 100 μM. An increase of Ni²⁺ in the sample to 200 μM increased the cardiac contraction strength, on average, by 41%. The negative inotropic action of verapamil was essentially reduced by 100 μM Ni²⁺. Adrenaline added to the sample after Ni²⁺ produced stimulating effect on the cardiac muscle, with an almost twofold rise of the contraction amplitude. ACh (0.2 μM) decreased the cardiac contraction amplitude, on average, by 56.3%, whereas Ni²⁺ (200 μM) administered after ACh not only restored, but also stimulated partly the myocardial work. Within several parts of percent there was an increase of such isometric contraction parameters as amplitude of the effort developed by muscle, maximal rate, maximal acceleration, time of semirise and semifall. The obtained experimental results indicate that the functional activity of the frog pacemaker and contractile cardiomyocytes is regulated by Ca²⁺-dependent mechanisms. Structure of these mechanisms includes the potential-controlled Land T-channels of the plasma membrane as well as Na⁺,Ca²-exchangers characteristic exclusively of contractile cardiomyocytes. The existence of these differences seems to be due to the cardiomyocyte morphological peculiarities that appeared in evolution at the stage of the functional cell specialization.     

Intracellular Ca<Superscript>2+</Superscript> Pools and Fluxes in Cardiac Muscle-Derived H9c2 Cells

Relevant Ca²⁺ pools and fluxes in H9c2 cells have been studied using fluorescent indicators and Ca²⁺-mobilizing agents. Vasopressin produced a cytoplasmic Ca²⁺ peak with half-maximal effective concentration of 6 nM, whereas thapsigargin-induced Ca²⁺ increase showed half-maximal effect at 3 nM. Depolarization of the mitochondrial inner membrane by protonophore was also associated with an increase in cytoplasmic Ca²⁺. Ionomycin induced a small and sustained depolarization, while thapsigargin had a small but transient effect. The thapsigargin-sensitive Ca²⁺ pool was also sensitive to ionomycin, whereas the protonophore-sensitive Ca²⁺ pool was not. The vasopressin-induced cytoplasmic Ca²⁺ signal, which caused a reversible discharge of the sarco-endoplasmic reticulum Ca²⁺ pool, was sensed as a mitochondrial Ca²⁺ peak but was unaffected by the permeability transition pore inhibitor cyclosporin A. The mitochondrial Ca²⁺ peak was affected by cyclosporin A when the Ca²⁺ signal was induced by irreversible discharge of the intracellular Ca²⁺ pool, i.e., adding thapsigargin. These observations indicate that the mitochondria interpret the cytoplasmic Ca²⁺ signals generated in the reticular store.     

Ca2+-dependent regulatory activity of recoverin in photoreceptor raft structures: The role of caveolin-1

Recoverin is a Ca²⁺-binding protein implicated in the Ca²⁺-dependent regulation of desensitization of visual receptor rhodopsin in vertebrate retinal rods. Here we report that Ca²⁺ sensitivity of recoverin regulating rhodopsin phosphorylation increases in the presence of the photoreceptor membranes enriched in raft structures. The observed effect is mediated by a key protein component of raft structures caveolin-1. The presence of recombinant fragment Phe⁸¹-Arg¹⁰¹ of the caveolin-1 cytoplasmic domain enhances Ca²⁺ affinity of recoverin, therefore affecting its Ca²⁺-dependent regulatory activity.     

Role of matrix metalloprotease-2 in oxidant activation of Ca<Superscript>2+</Superscript>ATPase by hydrogen peroxide in pulmonary vascular smooth muscle plasma membrane

Exposure of bovine pulmonary artery smooth muscle plasma membrane suspension with the oxidant H₂O₂ (1 mM) stimulated Ca²⁺ATPase activity. We sought to determine the role of matrix metalloprotease-2 (MMP-2) in stimulating Ca²⁺ATPase activity by H₂O₂ in the smooth muscle plasma membrane. The smooth muscle membrane possesses a Ca²⁺-dependent protease activity in the gelatin containing zymogram having an apparent molecular mass of 72 kDa. The 72 kDa protease activity was found to be inhibited by EGTA, 1: 10-phenanthroline, a2-macroglobulin and tissue inhibitor of metalloprotease-2 (TIMP-2) indicating that the Ca²⁺-dependent 72 kDa protease is the MMP-2. Western immunoblot studies of the membrane suspension with polyclonal antibodies of MMP-2 and TIMP-2 revealed that MMP-2 and TIMP-2, respectively, are the ambient matrix metalloprotease and the corresponding tissue inhibitor of metalloprotease in the membrane. In addition to increasing the Ca²⁺ATPase activity, H₂O₂ also enhanced the activity of the smooth muscle plasma membrane associated protease activity as evidenced by its ability to degrade¹⁴C-gelatin. The protease activity and the Ca²⁺ATPase activity were prevented by the antioxidant, vitamin E, indicating that the effect produced by H₂O₂ was due to reactive oxidant species(es). Both basal and H₂O₂ stimulated MMP-2 activity and Ca²⁺ATPase activity were inhibited by the general inhibitors of matrix metalloproteases: EGTA, 1: 10-phenanthroline, α₂-macroglobulin and also by TIMP-2 (the specific inhibitor of MMP-2) indicating that H₂O₂ increased MMP-2 activity and that subsequently stimulated Ca²⁺ATPase activity in the plasma membrane. This was further confirmed by the following observations: (i) adding low doses of MMP-2 or H₂O₂ to the smooth muscle membrane suspension caused submaximal increase in Ca²⁺ATPase activity, and pretreatment with TIMP-2 prevents the increase in Ca²⁺ATPase activity; (ii) combined treatment of the membrane with low doses of MMP-2 and H₂O₂ augments further the Ca²⁺ATPase activity caused by the respective low doses of either H₂O₂ or MMP-2; and (iii) pretreatment with TIMP-2 prevents the increase in Ca²⁺ATPase activity in the membrane caused by the combined treatment of MMP-2 and H₂O₂.     

Store-Dependent Entry of Ca<Superscript>2+</Superscript> into Sensory Neurons of the Rat

We studied store-dependent (activated by depletion of the endoplasmic reticulum, ER, store) entry of Ca²⁺ from the extracellular medium into neurons of the rat spinal ganglia (small- and medium-sized cells; diameter, 18 to 36 μm). Activation of ryanodine-sensitive receptors of the ER in the studied neurons superfused by Tyrode solutions containing Ca²⁺ or with no Ca²⁺ was provided by application of 10 mM caffeine. The decay phase of caffeine-induced calcium transients in a Ca²⁺-containing solution was significantly longer than that in a Ca²⁺-free solution. This fact allows us to suppose that such a phenomenon is determined by Ca²⁺ entry into the neuron from the extracellular medium activated by caffeine-induced depletion of the ER store. Substitution of Ca²⁺-free extracellular solution by Ca²⁺-containing Tyrode solution, after depletion of the ER stores induced by applications of 100 nM ryanodine, 200 μM ATP, or 1 μM thapsigargin, resulted in increases in the concentration of intracellular Ca²⁺. These observations allow us to postulate that store-dependent Ca²⁺ entry into the studied neurons is activated after depletion not only of the inositol trisphosphate-sensitive ER store but also of the ryanodine-sensitive store. This entry also occurs after blocking of ATPases of the ER by thapsigargin. The kinetic characteristics of the rising phase of store-dependent Ca²⁺ entry induced by depletion of the ER stores under the influence of various agents are dissimilar; this can be related to different mechanisms of activation of such signals and/or to a compartmental organization of the ER. Neirofiziologiya/Neurophysiology, Vol. 37, No. 3, pp. 277–283, May–June, 2005.     

Ca<Superscript>2+</Superscript> Buffering Capacity of Mitochondria After Oxygen-Glucose Deprivation in Hippocampal Neurons

In an earlier study, we showed that mitochondria hyperpolarized after short periods of oxygen-glucose deprivation (OGD), and this response appeared to be associated with subsequent apoptosis or survival. Here, we demonstrated that hyperpolarization following short periods of OGD (30 min; 30OGD group) increased the cytosolic Ca²⁺ ([Ca²⁺]_c) buffering capacity in mitochondria. After graded OGD (0 min (control), 30 min, 120 min), rat cultured hippocampal neurons were exposed to glutamate, evoking Ca²⁺influx. The [Ca²⁺]_c level increased sharply, followed by a rapid increase in mitochondrial Ca²⁺ [Ca²⁺]_m. The increase in the [Ca²⁺]_m level accompanied a reduction in the [Ca²⁺]_c level. After reaching a peak, the [Ca²⁺]_c level decreased more rapidly in the 30OGD group than in the control group. This buffering reaction was pronounced in the 30OGD group, but not in the 120OGD group. The enhanced buffering capacity of the mitochondria may be linked to preconditioning after short-term ischemic episodes.     

Conformational dynamics of recoverin's Ca2+-myristoyl switch probed by 15N NMR relaxation dispersion and chemical shift analysis

Recoverin, a member of the neuronal calcium sensor (NCS) branch of the calmodulin superfamily, serves as a calcium sensor in retinal rod cells. Ca²⁺‐induced conformational changes in recoverin promote extrusion of its covalently attached myristate, known as the Ca²⁺‐myristoyl switch. Here, we present nuclear magnetic resonance (NMR) relaxation dispersion and chemical shift analysis on ¹⁵N‐labeled recoverin to probe main chain conformational dynamics. ¹⁵N NMR relaxation data suggest that Ca²⁺‐free recoverin undergoes millisecond conformational dynamics at particular amide sites throughout the protein. The addition of trace Ca²⁺ levels (0.05 equivalents) increases the number of residues that show detectable relaxation dispersion. The Ca²⁺‐dependent chemical shifts and relaxation dispersion suggest that recoverin has an intermediate conformational state (I) between the sequestered apo state (T) and Ca²⁺ saturated extruded state (R): T ? I ? R. The first step is a fast conformational equilibrium ([T]/[I] < 100) on the millisecond time scale (τ_exδω < 1). The final step (I ? R) is much slower (τ_exδω > 1). The main chain structure of I is similar in part to the structure of half‐saturated E85Q recoverin with a sequestered myristoyl group. We propose that millisecond dynamics during T ? I may transiently increase the exposure of Ca²⁺‐binding sites to initiate Ca²⁺ binding that drives extrusion of the myristoyl group during I ? R. Proteins 2011; © 2011 Wiley‐Liss, Inc.     

Effects of Copper on T-Type Ca<Superscript>2+</Superscript> Channels in Mouse Spermatogenic Cells

Low voltage-activated, rapidly inactivating T-type Ca²⁺ channels are found in a variety of cells, where they regulate electrical activity and Ca²⁺ entry. In whole-cell patch-clamp recordings from mouse spermatogenic cells, trace element copper (Cu²⁺) inhibited T-type Ca²⁺ current (I _T-Ca) with IC₅₀ of 12.06 μM. Inhibition of I _T-Ca by Cu²⁺ was concentration-dependent and mildly voltage-dependent. When voltage stepped to −20 mV, Cu²⁺ (10 μM) inhibited I _T-Ca by 49.6 ± 4.1%. Inhibition of I _T-Ca by Cu²⁺ was accompanied by a shift of −2.23 mV in the voltage dependence of steady-state inactivation. Cu²⁺ upshifted the current–voltage (I-V) curve. To know the change of the gating kinetics of T-type Ca²⁺ channels, we analyzed the effect of Cu²⁺ on activation, inactivation, deactivation and reactivation of T-type Ca²⁺ channels. Since T-type Ca²⁺ channels are a key component in capacitation and the acrosome reaction, our data suggest that Cu²⁺ can affect male reproductive function through T-type Ca²⁺ channels as a preconception contraceptive material.