Temperature Dependence Of Ca2 Research Articles

Temperature Dependence of Fusion Kinetics and Fusion Pores in Ca2+-triggered Exocytosis from PC12 Cells

The temperature dependence of Ca2+-triggered exocytosis was studied using carbon fiber amperometry to record the release of norepinephrine from PC12 cells. Single-vesicle fusion events were examined at temperatures varying from 12 to 28°C, and with release elicited by depolarization. Measurements were made of the initial and maximum frequencies of exocytotic events, of fusion pore lifetime, flux through the open fusion pore, kiss-and-run versus full-fusion probability, and parameters associated with the shapes of amperometric spikes. The fusion pore open-state flux, and all parameters associated with spike shape, including area, rise time, and decay time, had weak temperature dependences and activation energies in the range expected for bulk diffusion in an aqueous solution. Kiss-and-run events also varied with temperature, with lower temperatures increasing the relative probability of kiss-and-run events by ∼50%. By contrast, kinetic parameters relating to the frequency of exocytotic events and fusion pore transitions depended much more strongly on temperature, suggesting that these processes entail structural rearrangements of proteins or lipids or both. The weak temperature dependence of spike shape suggests that after the fusion pore has started to expand, structural transitions of membrane components are no longer kinetically limiting. This indicates that the content of a vesicle is expelled completely after fusion pore expansion.

Role of calcium ions in the structure and function of thedi-isopropylfluorophosphatase from Loligo vulgaris

Di-isopropylfluorophosphatase (DFPase) is shown to contain two high-affinity Ca2+-binding sites, which are required for catalytic activity and stability. Incubation with chelating agents results in the irreversible inactivation of DFPase. From titrations with Quin 2 [2-({2-[bis(carboxymethyl)amino]-5-methylphenoxy}-methyl)-6-methoxy-8-[bis(carboxymethyl)-amino]quinoline], a lower-affinity site with dissociation constants of 21 and 840nM in the absence and the presence of 150mM KCl respectively was calculated. The higher-affinity site was not accessible, indicating a dissociation constant of less than 5.3nM. Stopped-flow experiments have shown that the dissociation of bound Ca2+ occurs in two phases, with rates of approx. 1.1 and 0.026s-1 corresponding to the dissociation from the low-affinity and high-affinity sites respectively. Dissociation rates depend strongly on temperature but not on ionic strength, indicating that Ca2+ dissociation is connected with conformational changes. Limited proteolysis, CD spectroscopy, dynamic light scattering and the binding of 8-anilino-1-naphthalenesulphonic acid have been combined to give a detailed picture of the conformational changes induced on the removal of Ca2+ from DFPase. The Ca2+ dissociation is shown to result in a primary, at least partly reversible, step characterized by a large decrease in DFPase activity and some changes in enzyme structure and shape. This step is followed by an irreversible denaturation and aggregation of the apo-enzyme. From the temperature dependence of Ca2+ dissociation and the denaturation results we conclude that the higher-affinity Ca2+ site is required for stabilizing DFPase's structure, whereas the lower-affinity site is likely to fulfil a catalytic function.

Role of calcium ions in the structure and function of the di-isopropylfluorophosphatase from Loligo vulgaris.

Di-isopropylfluorophosphatase (DFPase) is shown to contain two high-affinity Ca(2+)-binding sites, which are required for catalytic activity and stability. Incubation with chelating agents results in the irreversible inactivation of DFPase. From titrations with Quin 2 [2-([2-[bis(carboxymethyl)amino]-5-methylphenoxy]-methyl)-6-methoxy-8-[bis(carboxymethyl)-amino]quinoline], a lower-affinity site with dissociation constants of 21 and 840 nM in the absence and the presence of 150 mM KCl respectively was calculated. The higher-affinity site was not accessible, indicating a dissociation constant of less than 5.3 nM. Stopped-flow experiments have shown that the dissociation of bound Ca(2+) occurs in two phases, with rates of approx. 1.1 and 0.026 s(-1) corresponding to the dissociation from the low-affinity and high-affinity sites respectively. Dissociation rates depend strongly on temperature but not on ionic strength, indicating that Ca(2+) dissociation is connected with conformational changes. Limited proteolysis, CD spectroscopy, dynamic light scattering and the binding of 8-anilino-1-naphthalenesulphonic acid have been combined to give a detailed picture of the conformational changes induced on the removal of Ca(2+) from DFPase. The Ca(2+) dissociation is shown to result in a primary, at least partly reversible, step characterized by a large decrease in DFPase activity and some changes in enzyme structure and shape. This step is followed by an irreversible denaturation and aggregation of the apo-enzyme. From the temperature dependence of Ca(2+) dissociation and the denaturation results we conclude that the higher-affinity Ca(2+) site is required for stabilizing DFPase's structure, whereas the lower-affinity site is likely to fulfil a catalytic function.

Temperature dependence of Ca2+ wave properties in cardiomyocytes: implications for the mechanism of autocatalytic Ca2+ release in wave propagation

Digital imaging microscopy of fluo-3 fluorescence was used to study the velocity and shape of intracellular Ca2+ waves in isolated rat cardiomyocytes as a function of temperature. Decreasing the temperature from 37 to 17 degrees C reduced the longitudinal wave velocity by a factor of 1.8 and remarkably slowed the decay of [Ca2+]i in the trailing flank of a wave. Using image analysis, rise times, and half-maximum decay times of local Ca2+ transients, which characterize the processes of local Ca2+ release and removal, were determined as a function of temperature. Apparent activation energies for wave front propagation, local Ca2+ release, and local Ca2+ removal were derived from Arrhenius plots and amounted to -23, -28, and -46 kJ/mol, respectively. The high activation energy of Ca2+ removal, which arises from the activity of the sarcoplasmic reticulum (SR) Ca2+ ATPase, relative to those of longitudinal wave propagation and local Ca2+ release excludes the hypothetical mechanism of regenerative "spontaneous Ca2+ release," in which Ca2+ that has been taken up from the approaching wavefront triggers Ca2+ release at a luminal site of the SR. It is consistent, however, with the hypothesis that Ca2+ wave propagation is based on Ca(2+)-induced Ca2+ release where Ca2+ triggers release on the cytosolic face of the SR.

Temperature-dependent calcium sensitivity changes in skinned muscle fibres of rat and toad.

Single mechanically skinned muscle fibres of different types (fast- and slow-twitch mammalian; slow and twitch amphibian) were successively activated in solutions of various Ca2+ concentrations at different temperatures. An increase in temperature from 5 to 22 degrees C reversibly shifted the isometric steady-state force-pCa curves towards higher Ca2+ concentration for individual fibres of each of the muscle types. A further increase in temperature to 35 degrees C in mammalian fibres resulted in an additional decrease in Ca2+ sensitivity. The temperature dependence of Ca2+ sensitivity was greater in the 'faster' fibre types: fast-twitch greater than slow-twitch; twitch greater than slow. The maximum isometric force response, P0, of both rat and toad skinned fibres was found to be strongly dependent on temperature below 22 degrees C. No detectable force could be induced by Ca2+ in mammalian muscle fibres at 0-1 degree C while in toad fibres P0 decreased by about 90% when temperature dropped from 20 to 0 degree C. Since in mechanically skinned fibres of other amphibians (Bufo bufo, Rana species) P0 is only marginally affected it is likely that the P0-temperature relations are indicative of the range of temperature over which the muscles are normally functional. The P0-temperature relations of skinned muscle fibres closely resembled the P0-temperature relations of tetanically stimulated intact muscle preparations from the same species of animals suggesting that the contractile apparatus is mainly responsible for the variation of force response with temperature in intact muscle.

Role of phospholipids in the calcium-dependent ATPase of the sarcoplasmic reticulum. Enzymatic and ESR studies with phospholipid-replaced membranes.

Three types of partially purified ATPase enzymes having different phospholipid contents and compositions have been prepared: (a) an enzyme whose phospholipid moiety has been replaced predominantly by dioleoyl lecithin (DOL-enzyme), with about the same phospholipid content as the original sarcoplasmic reticulum, (b) dipalmitoyl lecithin-replaced enzyme whose phospholipid content is 30% of that of DOL-enzyme (DPL-enzyme), and (c) a partially delipidated enzyme with about the same phospholipid content as DPL-enzyme but with the original sarcoplasmic reticulum phospholipid composition (del-enzyme). The temperature dependence of Ca2+-activated ATPase activity of these preparations showed clearcut differences; with DOL-enzyme there was no appreciable break in the Arrhenius plot in the 3-40 degrees range; DPL-enzyme showed a break at 29 degrees, and del-enzyme and sarcoplasmic reticulum one at 18 degrees. Transition temperatures obtained from ESR studies with the use of spin-labeled stearic acid incorporated into the membranes agreed with those derived from ATPase assays. Thermo-dynamic analysis of the ATP hydrolysis rates shows that DPL-enzyme has considerably larger values of activation enthalpy and activation entropy below the transition temperature (29 degrees) than those of the other preparations, while all enzyme preparations show similar free energies of activation. The ESR data show that below their transition temperatures DPL-enzyme, and to a lesser degree del-enzyme, have a strongly restricted motion of their phospholipid molecules as compared with either DOL-enzyme or sarcoplasmic reticulum. Studies on the formation and decomposition of phosphoenzyme have been carried out with the three types of ATPase preparations. At 0 degrees, the rate of inorganic phosphate liberation is 8 times lower in DPL-enzyme than in del-enzyme with little difference in the steady state level of phosphoenzyme. In DOL-enzyme, the level of phosphoenzyme and the rate of inorganic phosphate liberation are 1.8 and 3.5 times higher than the corresponding values obtained with del-enzyme. Addition of ADP to the phosphorylated intermediate of DPL-enzyme induces a fast reversal of the phosphorylation reaction. These results indicate that the physical state of the phospholipid molecules associated with the enzyme affects the decomposition of phosphoenzyme, with little effect on the phosphorylation reaction and its reversal.