Calcium binding and allosteric signaling mechanisms for the sarcoplasmic reticulum Ca(2+) ATPase

The sarcoplasmic reticulum Ca(2+) ATPase (SERCA) is a membrane-bound pump that utilizes ATP to drive calcium ions from the myocyte cytosol against the higher calcium concentration in the sarcoplasmic reticulum. Conformational transitions associated with Ca(2+) -binding are important to its catalytic function. We have identified collective motions that partition SERCA crystallographic structures into multiple catalytically-distinct states using principal component analysis. Using Brownian dynamics simulations, we demonstrate the important contribution of surface-exposed, polar residues in the diffusional encounter of Ca(2+) . Molecular dynamics simulations indicate the role of Glu309 gating in binding Ca(2+) , as well as subsequent changes in the dynamics of SERCA's cytosolic domains. Together these data provide structural and dynamical insights into a multistep process involving Ca(2+) binding and catalytic transitions.     

Phosphatidylinositol 4,5-biphosphate stimulates parotid endoplasmic reticulum (Ca2+ + Mg2+)-ATP'ase

The effect of various phospholipids on the (Ca2+ + Mg2+)-ATP'ase of rat parotid endoplasmic reticulum was investigated. At a concentration of 20 micrograms/ml, phosphatidylinositol 4,5-biphosphate stimulated the Ca2+-activated ATP'ase by 69%. None of the other phospholipids tested or 10 microM inositol 1,4,5-triphosphate had any significant effect on the ATP'ase. Stimulation by phosphatidylinositol 4,5-biphosphate was concentration dependent and half maximal stimulation required 8 micrograms/ml of the phospholipid. The results suggest that changes in the cellular concentration of phosphatidylinositol 4,5-biphosphate may be of importance in regulating Ca2+ transport by the endoplasmic reticulum.     

Anion dependence of Ca2+ transport and (Ca2+ + K+)-stimulated Mg2+-dependent transport ATPase in rat pancreatic endoplasmic reticulum

Anion dependence of (Ca2+ + K+)-stimulated Mg2+-dependent transport ATPase and its phosphorylated intermediate have been characterized in both "intact" and "broken" vesicles from endoplasmic reticulum of rat pancreatic acinar cells using adenosine 5'-[gamma-32P] triphosphate ([gamma-32P]ATP). In intact vesicles (Ca2+ + K+)-Mg2+-ATPase activity was higher in the presence of Cl- or Br- as compared to NO3-, SCN-, cyclamate-, SO4(2-) or SO3(2-). Incorporation of 32P from [gamma-32P]ATP into the 100-kDa intermediate of this Ca2+ATPase was also higher in the presence of Cl-, Br-, NO3- or SCN- as compared to cyclamate-, SO4(2-) or SO3(2-). When the membrane permeability barrier to anions was abolished by breaking vesicle membrane with the detergent Triton X-100 (0.015%) (Ca2+ + K+)-Mg2+ATPase activity in the presence of weakly permeant anions, such as SO4(2-) and cyclamate-, increased to the level obtained with Cl-. However, 32P incorporation into 100-kDa protein was still higher in the presence of Cl- as compared to cyclamate-, indicating a direct effect of Cl- on the Ca2+ATPase molecule. The anion transport blocker 4,4-diisothiocyanostilbene-2,2-disulfonate (DIDS) inhibited (Ca2+ + K+)-Mg2+ATPase activity to about 10% of the Cl- stimulation level, irrespective of the sort of anions present in both intact and broken vesicles. This indicates a direct effect of DIDS on (Ca2+ + K+)-Mg2+ATPase. K+ ionophore valinomycin influenced (Ca2+ + K+)-Mg2+ATPase activity according to the actual K+ gradient: Ko+ greater than Ki+ caused inhibition, Ko+ less than Ki+ caused stimulation. From these results we conclude that Ca2+ transport into endoplasmic reticulum is coupled to ion movements which must occur to maintain electroneutrality.     

A non-specific Ca2+ (or Mg2+)-stimulated ATPase in rat heart sarcoplasmic reticulum

ATPase activity in rat heart sarcoplasmic reticulum was stimulated in a concentration-dependent manner by both Ca²⁺ and Mg²⁺ in the complete absence of the other cation. Increasing concentrations of Mg²⁺ produced an apparent inhibition of the Ca²⁺-dependent ATP hydrolysis. CDTA (trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetate) had no effect on these responses. The results indicate the presence of a low affinity non-specific divalent cation-stimulated ATPase in rat heart sarcoplasmic reticulum. However, sarcoplasmic reticulum vesicles transported Ca²⁺ with a high affinity (K_0.5 Ca²⁺ = 0.41 M) suggesting the presence of a high affinity Ca²⁺-transporting ATPase. Calmodulin did not stimulate rat heart sarcoplasmic reticulum ATPase activity over a range of Ca²⁺ and Mg²⁺ concentrations and failed to stimulate membrane phosphorylation and Ca²⁺ transport into sarcoplasmic reticulum vesicles. Calmodulin antagonists trifluoperazine and compound 48180 did not affect the ATPase activity. Catalytic subunit of cAMP-dependent protein kinase was also ineffective in stimulating the ATPase activity. These results suggest the presence of an ATPase activity in rat heart sarcoplasmic reticulum with different properties from the high affinity Ca²⁺-pumping ATPase previously characterized in dog heart and other species.Abbreviations cAMP adenosine 3,5-monophosphate - CaM calmodulin - CDTA trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetate - EDTA ethylene-diaminetetraacetate - EGTA ethylene glycol bis(-aminoethyl ether)-N,N,N,N-tetraacetate - PLB phospholamban - SR sarcoplasmic reticulum - TFP trifluoperazine     

Characterization of Mg2+- and (Ca2+ + Mg2+)-ATPase activity in adipocyte endoplasmic reticulum

The presence of an energy-dependent calcium uptake system in adipocyte endoplasmic reticulum (D. E. Bruns, J. M. McDonald, and L. Jarett, 1976, J. Biol. Chem.251, 7191–7197) suggested that this organelle might possess a calcium-stimulated transport ATPase. This report describes two types of ATPase activity in isolated microsomal vesicles: a nonspecific, divalent cation-stimulated ATPase (Mg²⁺-ATPase) of high specific activity, and a specific, calcium-dependent ATPase (Ca²⁺ + Mg²⁺-ATPase) of relatively low activity. Mg²⁺-ATPase activity was present in preparations of mitochondria and plasma membranes as well as microsomes, whereas the (Ca²⁺ + Mg²⁺)-ATPase activity appeared to be localized in the endoplasmic reticulum component of the microsomal fraction. Characterization of microsomal Mg²⁺-ATPase activity revealed apparent K_m values of 115 μm for ATP, 333 μm for magnesium, and 200 μm for calcium. Maximum Mg²⁺-ATPase activity was obtained with no added calcium and 1 mm magnesium. Potassium was found to inhibit Mg²⁺-ATPase activity at concentrations greater than 100 mm. The energy of activation was calculated from Arrhenius plots to be 8.6 kcal/mol. Maximum activity of microsomal (Ca²⁺ + Mg²⁺)-ATPase was 13.7 nmol ³²P/mg/min, which represented only 7% of the total ATPase activity. The enzyme was partially purified by treatment of the microsomes with 0.09% deoxycholic acid in 0.15 m KCl which increased the specific activity to 37.7 nmol ³²P/mg/min. Characterization of (Ca²⁺ + Mg²⁺)-ATPase activity in this preparation revealed a biphasic dependence on ATP with a Hill coefficient of 0.80. The apparent K_m^s for magnesium and calcium were 125 and 0.6–1.2 μm, respectively. (Ca²⁺ + Mg²⁺)-ATPase activity was stimulated by potassium with an apparent K_m of 10 mm and maximum activity reached at 100 mm potassium. The energy of activation was 21.5 kcal/mol. The kinetics and ionic requirements of (Ca²⁺ + Mg²⁺)-ATPase are similar to those of the (Ca²⁺ + Mg²⁺)-ATPase in sarcoplasmic reticulum. These results suggest that the (Ca²⁺ + Mg²⁺)-ATPase of adipocyte endoplasmic reticulum functions as a calcium transport enzyme.     

Phosphorylated intermediate of (Ca2+ + K+)-stimulated Mg2+-dependent transport ATPase in endoplasmic reticulum from rat pancreatic acinar cells

Formation and decomposition of the phosphorylated intermediate of endoplasmic reticulum (Ca2+ + Mg2+)-ATPase from pancreatic acinar cells have been studied using lithium dodecyl sulfate- and tetradecyltrimethylammonium bromide-polyacrylamide gel electrophoresis. Incorporation of 32P from [gamma-32P]ATP is Ca2+-dependent (approximate Km for free [Ca2+] = 2-3 X 10(-8) mol/liter). Formation of the 100-kDa phosphoprotein is rapid, reaching maximal 32Pi incorporation within 1 s at room temperature. At 4 degrees C, phosphorylation is slower and dephosphorylation is drastically decreased. For dephosphorylation, Mg2+ and monovalent cations such as K+ or Na+ are necessary. Vanadate inhibits both 32P incorporation and 32P liberation dose dependently (Km = 3 X 10(-6) mol/liter), whereas mitochondrial inhibitors and ouabain have no effect. The phosphoprotein is stable at pH 2 and destabilizes with increasing pH being completely decomposed at pH 9. Reduction of 32P incorporation in the presence of high concentrations of cold ATP and hydroxylamine suggests formation of acylphosphate present in the ATPase intermediate. The characteristics of Ca2+, cation, and pH dependencies of the ATPase activity are similar to those previously described for MgATP-dependent Ca2+ transport into rough endoplasmic reticulum from pancreatic acinar cells (Bayerd?rffer, E., Streb, H., Eckhardt, L., Haase, W., and Schulz, I. (1984) J. Membr. Biol. 81, 69-82). The data suggest that the 100-kDa phosphoprotein as described in this study is the intermediate of this Ca2+ transport ATPase.     

Contribution of endoplasmic reticulum to Ca(2+) signals in Dictyostelium depends on extracellular Ca(2+)

We recently reported the first molecular genetic evidence that Dictyostelium Ca²⁺ responses to chemoattractants include a contribution from the endoplasmic reticulum (ER) – responses are enhanced in mutants lacking calreticulin or calnexin, two major Ca²⁺-binding proteins in the ER, even though the influx of Ca²⁺ into the mutants is reduced. Compared with wild-type cells, the ER in the mutants contributes at least 30–70 nM additional Ca²⁺ to the responses. Here we report that this additional ER contribution to the cytosolic Ca²⁺ signal depends upon extracellular Ca²⁺– it does not occur in the absence of extracellular Ca²⁺, increases to a maximum as the extracellular Ca²⁺ levels rise to 10 μM and then remains constant at extracellular Ca²⁺ concentrations up to at least 250 μM. These results suggest that Ca²⁺ influx causes the intracellular release, in the simplest scenario by a mechanism involving Ca²⁺-induced Ca²⁺ release from the ER. By way of contrast, we show that Ca²⁺ responses to mechanical stimulation are reduced, but still occur in the absence of extracellular Ca²⁺. Unlike the responses to chemoattractants, mechanoresponses thus include contributions from the ER that are independent of extracellular Ca²⁺.     

Markers of squamous cell carcinoma in sarco/endoplasmic reticulum Ca ATPase 2 heterozygote mice keratinocytes

A mutation of Atp2a2 gene encoding the sarco/endoplasmic reticulum Ca²⁺-ATPase 2 (SERCA2) causes Darier's disease in human and null mutation in one copy of Atp2a2 leads to a high incidence of squamous cell tumor in a mouse model. In SERCA2 heterozygote (SERCA2^+/−) mice keratinocytes, mechanisms involved in partial depletion of SERCA2 gene and its related tumor induction have not been studied. In this study, we investigated Ca²⁺ signaling and differential gene expression in primary cultured keratinocytes from SERCA2^+/− mice. SERCA2^+/− keratinocytes showed reduced initial increases in intracellular concentration of calcium in response to ATP, a G-protein coupled receptor agonist, and higher store-operated Ca²⁺ entry with the treatment of thapsigargin, an inhibitor of SERCA, compared to wild type kerationcytes. Protein expressions of plasma membrane Ca²⁺ ATPases, NFATc1, phosphorylated ERK, JNK, and phospholipase γ1 were increased in SERCA2^+/− keratinocytes. Using the gene fishing system, we first found in SERCA2^+/− keratinocytes that gene level of tumor-associated calcium signal transducer 1, crystalline αB, procollagen XVIII α1, and nuclear factor I-B were increased. Expression of involucrin, a marker of keratinocyte differentiation, was decreased in SERCA2^+/− keratinocytes. These results suggest that the alterations of Ca²⁺ signaling by SERCA2 haploinsufficiency alternate the gene expression of tumor induction and differentiation in keratinocytes.     

Pilocarpine-induced status epilepticus causes N-methyl-D-aspartate receptor-dependent inhibition of microsomal Mg(2+)/Ca(2+) ATPase-mediated Ca(2+) uptake

Status epilepticus is associated with sustained and elevated levels of cytosolic Ca(2+). To elucidate the mechanisms associated with changes of cytosolic Ca(2+) after status epilepticus, this study was initiated to evaluate the effect of pilocarpine-induced status epilepticus on Mg(2+)/Ca(2+) ATPase-mediated Ca(2+) uptake in microsomes isolated from rat cortex, because the Ca(2+) uptake mechanism plays a major role in regulating intracellular Ca(2+) levels. The data demonstrated that the initial rate and overall Ca(2+) uptake in microsomes from pilocarpine treated animals were significantly inhibited compared with those in microsomes from saline-treated control animals. It was also shown that the inhibition of Ca(2+) uptake caused by status epilepticus was not an artifact of increased Ca(2+) release from microsomes, selective isolation of damaged microsomes from the homogenate, or decreased Mg(2+)/Ca(2+) ATPase protein in the microsomes. Pretreatment with the NMDA antagonist dizocilpine maleate blocked status epilepticus-induced inhibition of the initial rate and overall Ca(2+) uptake. The data suggest that inhibition of microsomal Mg(2+)/Ca(2+) ATPase Ca(2+) uptake is involved in NMDA-dependent deregulation of cytosolic Ca(2+) homeostasis associated with status epilepticus.     

Characterization of a Listeria monocytogenes Ca(2+) pump: a SERCA-type ATPase with only one Ca(2+)-binding site

We have characterized a putative Ca(2+)-ATPase from the pathogenic bacterium Listeria monocytogenes with the locus tag lmo0841. The purified and detergent-solubilized protein, which we have named Listeria monocytogenes Ca(2+)-ATPase 1 (LMCA1), performs a Ca(2+)-dependent ATP hydrolysis and actively transports Ca(2+) after reconstitution in dioleoylphosphatidyl-choline vesicles. Despite a high sequence similarity to the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1a) and plasma membrane Ca(2+)-ATPase (PMCA), LMCA1 exhibits important biochemical differences such as a low Ca(2+) affinity (K(0.5) ～80 μm) and a high pH optimum (pH ～9). Mutational studies indicate that the unusually high pH optimum can be partially ascribed to the presence of an arginine residue (Arg-795), corresponding in sequence alignments to the Glu-908 position at Ca(2+) binding site I of rabbit SERCA1a, but probably with an exposed position in LMCA1. The arginine is characteristic of a large group of putative bacterial Ca(2+)-ATPases. Moreover, we demonstrate that H(+) is countertransported with a transport stoichiometry of 1 Ca(2+) out and 1 H(+) in per ATP hydrolyzed. The ATPase may serve an important function by removing Ca(2+) from the microorganism in environmental conditions when e.g. stressed by high Ca(2+) and alkaline pH.     

The mechanism of inhibition of the sarco/endoplasmic reticulum Ca2+ ATPase by paxilline

Although cis-diamminedichloroplatinum (II) (cisplatin) is a potent anticancer drug, clinical use of this agent is highly limited predominantly because of its strong side effects on the kidney and gastrointestinal tracts. We found that cisplatin impaired respiratory function and DNA of mitochondria in renal proximal tubules and small intestinal mucosal cells, thereby inducing apoptosis of epithelial cells. Cisplatin-induced mitochondrial dysfunction and DNA (mtDNA) injury, lipid peroxidation, and apoptosis of epithelial cells in the kidney and small intestine were strongly inhibited by L-carnitine. However, carnitine had no appreciable effect on the tumoricidal action of cisplatin against cancer cells inoculated in the peritoneal cavity. These results indicate that L-carnitine may have therapeutic potential for inhibiting the side effects of cisplatin and other anticancer agents in the kidney and small intestine.     

cGMP stimulates endoplasmic reticulum Ca(2+)-ATPase in vascular endothelial cells

Calcium is a crucial regulator of many physiological processes such as cell growth, division, differentiation, cell death and apoptosis. In this study, we examined the effect of cGMP on agonist-induced [Ca(2+)](i) transient in isolated rat aortic endothelial cells. 100 microM ATP was applied to the cells bathed in a Ca(2+)-free physiological solution to induce a [Ca(2+)](i) transient that was caused by Ca(2+) release from intracellular stores. cGMP, which was applied after [Ca(2+)](i) reached its peak level, accelerated the falling phase of [Ca(2+)](i) transient. Pre-treatment of the cells with CPA abolished the accelerating effect of cGMP on the falling phase of [Ca(2+)](i) transient. The effect of cGMP was reversed by KT5823, a highly specific inhibitor of protein kinase G. Taken together, these data suggest that cGMP may reduce [Ca(2+)](i) level by promoting Ca(2+) uptake through sarcoplasmic/endoplasmic reticulum ATPase and that the effect of cGMP may be mediated by protein kinase G.     

Beta-adrenergic potentiation of endoplasmic reticulum Ca(2+) release in brown fat cells

We find that the adrenergic agonist isoproterenol increases intracellular Ca(2+) concentration ([Ca(2+)](i)) in cultured rat brown adipocytes. At the concentration used (10 microM), isoproterenol-induced Ca(2+) responses were sensitive to block by either alpha(1)- or beta-adrenergic antagonists, suggesting an interaction between these receptor subtypes. Despite reliance on beta-adrenoceptor activation, the Ca(2+) response was not due solely to increases in cAMP because, administered alone, the selective beta(3)-adrenergic agonist BRL-37344 or forskolin did not increase [Ca(2+)](i). However, increased cAMP elicited vigorous [Ca(2+)](i) increases in the presence of barely active concentrations of the alpha-adrenergic agonist phenylephrine or the P2Y receptor agonist UTP. Consistent with isoproterenol recruiting only inositol 1,4,5-trisphosphate (IP(3))-sensitive Ca(2+) stores, endoplasmic reticulum store depletion by thapsigargin blocked isoproterenol-induced Ca(2+) increases, but removal of external Ca(2+) did not. These results argue that increases in cAMP sensitize the IP(3)-mediated Ca(2+) release system in brown adipocytes.     

Ca(2+) dynamics in the lumen of the endoplasmic reticulum in sensory neurons: direct visualization of Ca(2+)-induced Ca(2+) release triggered by physiological Ca(2+) entry

In cultured rat dorsal root ganglia neurons, we measured membrane currents, using the patch-clamp whole-cell technique, and the concentrations of free Ca(2+) in the cytosol ([Ca(2+)](i)) and in the lumen of the endoplasmic reticulum (ER) ([Ca(2+)](L)), using high- (Fluo-3) and low- (Mag-Fura-2) affinity Ca(2+)-sensitive fluorescent probes and video imaging. Resting [Ca(2+)](L) concentration varied between 60 and 270 microM. Activation of ryanodine receptors by caffeine triggered a rapid fall in [Ca(2+)](L) levels, which amounted to only 40--50% of the resting [Ca(2+)](L) value. Using electrophysiological depolarization, we directly demonstrate the process of Ca(2+)-induced Ca(2+) release triggered by Ca(2+) entry through voltage-gated Ca(2+) channels. The amplitude of Ca(2+) release from the ER lumen was linearly dependent on I(Ca).     

The endoplasmic reticulum as one continuous Ca(2+) pool: visualization of rapid Ca(2+) movements and equilibration

We investigated whether the endoplasmic reticulum (ER) is a functionally connected Ca(2+) store or is composed of separate subunits by monitoring movements of Ca(2+) and small fluorescent probes in the ER lumen of pancreatic acinar cells, using confocal microscopy, local bleaching and uncaging. We observed rapid movements and equilibration of Ca(2+) and the probes. The bulk of the ER at the base was not connected to the granules in the apical part, but diffusion into small apical ER extensions occurred. The connectivity of the ER Ca(2+) store was robust, since even supramaximal acetylcholine (ACh) stimulation for 30 min did not result in functional fragmentation. ACh could elicit a uniform decrease in the ER Ca(2+) concentration throughout the cell, but repetitive cytosolic Ca(2+) spikes, induced by a low ACh concentration, hardly reduced the ER Ca(2+) level. We conclude that the ER is a functionally continuous unit, which enables efficient Ca(2+) liberation. Ca(2+) released from the apical ER terminals is quickly replenished from the bulk of the rough ER at the base.     

Calmodulin-mediated regulation of calcium transport and (Ca2+ + Mg2+)-activated ATPase activity in isolated cardiac sarcoplasmic reticulum

A severalfold activation of calcium transport and (Ca2+ + Mg2+)-activated ATPase activity by micromolar concentrations of calmodulin was observed in sarcoplasmic reticulum vesicles obtained from canine ventricles. This activation was seen in the presence of 120 mM KCl. The ratio of moles of calcium transported per mol of ATP hydrolyzed remained at about 0.75 when calcium transport and (Ca2+ + Mg2+)-activated ATPase activity were measured in the presence and absence of calmodulin. Thus, the efficiency of the calcium transport process did not change. Stimulation of calcium transport by calmodulin involves the phosphorylation of one or more proteins. The major 32P-labeled protein, as determined by sodium dodecyl sulfate slab gel electrophoresis, was the 22,000-dalton protein called phospholamban. The Ca2+ concentration dependency of calmodulin-stimulated microsomal phosphorylation corresponded to that of calmodulin-stimulated (Ca2+ + Mg2+)-activated ATPase activity. Proteins of 11,000 and 6,000 daltons and other proteins were labeled to a lesser extent. A similar phosphorylation pattern was obtained when microsomes were incubated with cAMP-dependent protein kinase and ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. Phosphorylation produced by added cAMP-dependent protein kinase and calmodulin was additive. These studies provided further evidence for Ca2+-dependent regulation of calcium transport by calmodulin in sarcoplasmic reticulum that could play a role in the beat-to-beat regulation of cardiac relaxation in the intact heart.     

Regulation of cardiac sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase

Summary The two high affinity calcium binding sites of the cardiac (Ca²⁺ + Mg²⁺)-ATPase have been identified with the use of Eu³⁺. Eu³⁺ competes for the two high affinity calcium sites on the enzyme. With the use of laser-pulsed fluorescent spectroscopy, the environment of the two sites appear to be heterogeneous and contain different numbers of H₂O molecules coordinated to the ion. The ion appears to be occluded even further in the presence of ATP. Using non-radiative energy transfer studies, we were able to estimate the distance between the two Ca²⁺ sites to be between 9.4 to 10.2 A in the presence of ATP. Finally, from the assumption that the calcium site must contain four carboxylic side chains to provide the 6–8 ligands needed to coordinate calcium, and based on our recently published data, we predict the peptidic backbone of the two sites.     

Ca(2+) and Mg(2+)/ATP independently trigger homotypic membrane fusion in gastric secretory membranes

Exocytic activation of gastric parietal cells represents a massive transformation. We studied a step in this process, homotypic fusion of H,K-ATPase-containing tubulovesicles, using R18 dequenching. Ca²⁺ and Mg²⁺/ATP each caused dramatic dequenching, reflecting a change in R18 distribution from 5% to 65–90% of the assay's membranes in 2.5 min. These stimuli also triggered fusion between tubulovesicles and liposomes. Independent confirmation that dequenching represented membrane fusion was established by separating tubulovesicle–liposome fusion products on density gradients. Only agents that trigger fusion allowed the transmembrane H,K-ATPase to move to low-density fractions along with R18. EC₅₀ for Ca²⁺-triggered fusion was 150 n m and for Mg²⁺/ATP-triggered fusion 1 m m , the latter having a Hill coefficient of 2.5. ATP-triggered fusion was specific for Mg²⁺/ATP, required ATP hydrolysis, and was insensitive to inhibition of NSF and/or H,K-ATPase. Fusion initiated by either trigger caused tubulovesicles to become resistant to subsequent challenge by either trigger. Ca²⁺-and Mg²⁺/ATP-triggered fusion required protein component(s) in tubulovesicles, though this was required in only one of the fusing membranes since tubulovesicles fused well with liposomes containing no proteins. Our data suggest that exocytosis in parietal cells is triggered by separate but interacting pathways and is regulated by self-inhibition.     

Ca(2+) measurements in skinned cardiac fibers: effects of Mg(2+) on Ca(2+) activation of force and fiber ATPase.

In contrast to previous studies, a new fluorescent method was used to accurately determine the Ca(2+) concentration in test solutions used to activate skinned rat cardiac cells. This method used the calcium green-2 fluorescent indicator, which is shown to change its fluorescence over the Ca(2+) range responsible for Ca(2+) activation of force and ATPase. The dissociation constant (K(d)) of calcium green-2 for Ca(2+) was determined for three different Mg(2+) concentrations in solutions similar to those used in the experiment. Increasing Mg(2+) concentration from 1.0 to 8.0 mM had no significant effect on the Ca(2+) sensitivity of either force or actomyosin ATPase activity, in contrast to previous reported studies on force. The ATPase activity was activated at lower Ca(2+) concentration than the force. The ratio (ATPase/force) is proportional to the dissociation rate of force-generating myosin cross bridges and decreased during Ca(2+) activation. These findings are consistent with the hypothesis that cardiac muscle contraction is activated by a single Ca(2+)-specific binding site on troponin C.