thapsigargin and aluminum-fluoride

We document here the intrinsic fluorescence and 45Ca2+ binding properties of putative "E2P-related" complexes of Ca2+-free ATPase with fluoride, formed in the presence of magnesium, aluminum, or beryllium. Intrinsic fluorescence measurements suggest that in the absence of inhibitors, the ATPase complex with beryllium fluoride (but not those with magnesium or aluminum fluoride) does constitute an appropriate analog of the "ADP-insensitive" phosphorylated form of Ca2+-ATPase, the so-called "E2P" state. 45Ca2+ binding measurements, performed in the presence of 100 mm KCl, 5 mm Mg2+, and 20% Me2SO at pH 8, demonstrate that this ATPase complex with beryllium fluoride (but again not those with magnesium or aluminum fluoride) has its Ca2+ binding sites accessible for rapid, low affinity (submillimolar) binding of Ca2+ from the luminal side of SR. In addition, we specifically demonstrate that in this E2P-like form of ATPase, the presence of thapsigargin, 2,5-di-tert-butyl-1,4-dihydroxybenzene, or cyclopiazonic acid prevents 45Ca2+ binding (i.e. presumably prevents opening of the 45Ca2+ binding sites on the SR luminal side). Since crystals of E2P-related forms of ATPase have up to now been described in the presence of thapsigargin only, these results suggest that crystallizing an inhibitor-free E2P-like form of ATPase (like its complex with beryllium fluoride) would be highly desirable, to unambiguously confirm previous predictions about the exit pathway from the ATPase transmembrane Ca2+ binding sites to the SR luminal medium.

Topics: Adenosine Triphosphatases; Aluminum; Aluminum Compounds; Animals; Beryllium; Binding Sites; Biochemistry; Biological Transport; Calcium; Calcium-Transporting ATPases; Cell Membrane; Enzyme Inhibitors; Fluorides; Hydrogen-Ion Concentration; Indoles; Ions; Magnesium; Microscopy, Fluorescence; Phosphorylation; Protein Binding; Protein Conformation; Rabbits; Sarcoplasmic Reticulum; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Thapsigargin; Time Factors; Tryptophan

The structural natures of stable analogues for the ADP-insensitive phosphoenzyme (E2P) of Ca(2+)-ATPase formed in sarcoplasmic reticulum vesicles, i.e. the enzymes with bound beryllium fluoride (BeF.E2), bound aluminum fluoride (AlF.E2), and bound magnesium fluoride (MgF.E2), were explored and compared with those of actual E2P formed from P(i) without Ca(2+). Changes in trinitrophenyl-AMP fluorescence revealed that the catalytic site is strongly hydrophobic in BeF.E2 as in E2P but hydrophilic in MgF.E2 and AlF.E2; yet, the three cytoplasmic domains are compactly organized in these states. Thapsigargin, which was shown in the crystal structure to fix the transmembrane helices and, thus, the postulated Ca(2+) release pathway to lumen in a closed state, largely reduced the tryptophan fluorescence in BeF.E2 as in E2P, but only very slightly (hence, the release pathway is likely closed without thapsigargin) in MgF.E2 and AlF.E2 as in dephosphorylated enzyme. Consistently, the completely suppressed Ca(2+)-ATPase activity in BeF-treated vesicles was rapidly restored in the presence of ionophore A23187 but not in its absence by incubation with Ca(2+) (over several millimolar concentrations) at pH 6, and, therefore, lumenal Ca(2+) is accessible to reactivate the enzyme. In contrast, no or only very slow restoration was observed with vesicles treated with MgF and AlF even with A23187. BeF.E2 thus has the features very similar to those characteristic of the E2P ground state, although AlF.E2 and MgF.E2 most likely mimic the transition or product state for the E2P hydrolysis, during which the hydrophobic nature around the phosphorylation site is lost and the Ca(2+) release pathway is closed. The change in hydrophobic nature is probably associated with the change in phosphate geometry from the covalently bound tetrahedral ground state (BeF(3)(-)) to trigonal bipyramidal transition state (AlF(3) or AlF(4)(-)) and further to tetrahedral product state (MgF(4)(2-)), and such change likely rearranges transmembrane helices to prevent access and leakage of lumenal Ca(2+).

Topics: Adenosine Diphosphate; Adenosine Monophosphate; Aluminum Compounds; Animals; Beryllium; Biochemical Phenomena; Biochemistry; Calcimycin; Calcium; Calcium-Transporting ATPases; Catalytic Domain; Cell Membrane; Chelating Agents; Crystallography, X-Ray; Cytoplasm; Dose-Response Relationship, Drug; Fluorides; Hydrogen-Ion Concentration; Hydrolysis; Magnesium Compounds; Models, Chemical; Muscle, Skeletal; Phosphorylation; Protein Binding; Protein Conformation; Protein Structure, Tertiary; Rabbits; Sarcoplasmic Reticulum; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Spectrometry, Fluorescence; Thapsigargin; Time Factors; Tryptophan; Vanadates

P-type ATPases extract energy by hydrolysis of adenosine triphosphate (ATP) in two steps, formation and breakdown of a covalent phosphoenzyme intermediate. This process drives active transport and countertransport of the cation pumps. We have determined the crystal structure of rabbit sarcoplasmic reticulum Ca2+ adenosine triphosphatase in complex with aluminum fluoride, which mimics the transition state of hydrolysis of the counterion-bound (protonated) phosphoenzyme. On the basis of structural analysis and biochemical data, we find this form to represent an occluded state of the proton counterions. Hydrolysis is catalyzed by the conserved Thr-Gly-Glu-Ser motif, and it exploits an associative nucleophilic reaction mechanism of the same type as phosphoryl transfer from ATP. On this basis, we propose a general mechanism of occluded transition states of Ca2+ transport and H+ countertransport coupled to phosphorylation and dephosphorylation, respectively.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Aluminum Compounds; Amino Acid Motifs; Animals; Binding Sites; Biological Transport, Active; Calcium; Calcium-Transporting ATPases; Chemical Phenomena; Chemistry, Physical; Crystallization; Crystallography, X-Ray; Cytoplasm; Fluorides; Hydrolysis; Ion Transport; Models, Chemical; Models, Molecular; Phosphorylation; Protein Conformation; Protein Structure, Tertiary; Protons; Rabbits; Sarcoplasmic Reticulum; Thapsigargin; Thermodynamics

Studies presented here show that altering the intracellular redox balance by decreasing glutathione levels profoundly affects early signal transduction events in human T cells. In a T-cell receptor (TCR) signaling model, short-term pretreatment with buthionine sulfoximine, which specifically decreases intracellular glutathione, essentially abrogates the stimulation of calcium influx by anti-CD3 antibodies without significantly impairing other aspects of TCR-initiated signal transduction, such as overall levels of TCR-stimulated tyrosine phosphorylation. In an inflammatory-cytokine signaling model, the failure of tumor necrosis factor alpha to stimulate more than minimal tyrosine phosphorylation in lymphocytes is overcome by buthionine sulfoximine pretreatment--i.e., tumor necrosis factor alpha stimulates extensive tyrosine phosphorylation in glutathione-depleted lymphocytes. These redox-dependent changes in T-cell responsiveness suggest that the glutathione deficiency that we and others have demonstrated in human immunodeficiency virus-infected individuals may contribute significantly to the immunodeficiency and the increased inflammatory reactions in these individuals.

Topics: Aluminum Compounds; Calcium; CD3 Complex; Cell Line; Cytokines; Fluorides; Glutathione; Humans; In Vitro Techniques; Oxidation-Reduction; Phosphoserine; Phosphothreonine; Phosphotyrosine; Receptors, Antigen, T-Cell; Signal Transduction; T-Lymphocytes; Terpenes; Thapsigargin; Tyrosine

In the human submandibular ductal cell line (HSG) thapsigargin and carbachol stimulated Ca2+ release from the internal Ca2+ pool, resulting in the activation of capacitatively regulated Ca2+ entry (CRCE). This entry pathway was permeant to both Ca2+ and Mn2+, blocked by Ni2+ and insensitive to the muscarinic antagonist, atropine. Carbachol also stimulated an increase in cytosolic [Ca2+] in internal Ca(2+)-pool-depleted (i.e. thapsigargin-treated) cells which was dependent on the presence of external Ca2+ and blocked by Ni2+, demonstrating that it was due to Ca2+ entry. However, under the same experimental conditions, carbachol was unable to stimulate Mn2+ entry. Additionally, this latter carbachol-stimulated Ca2+ entry pathway was blocked by atropine. Pretreatment of HSG cells with AlF4-increased basal rates of Mn2+ entry due to CRCE activation, but attenuated carbachol-stimulated Ca2+ entry into thapsigargin-treated cells. The data suggest that two distinct divalent cation entry pathways are activated in muscarinic-receptor-stimulated HSG cells; a CRCE mechanism, permeable to both Mn2+ and Ca2+, and a second entry mechanism, permeable only to Ca2+. The latter does not depend on internal pool depletion, but appears to be regulated via G-protein activation.

Topics: Aluminum Compounds; Calcium; Carbachol; Cell Line; Fluorides; GTP-Binding Proteins; Humans; Nickel; Receptors, Muscarinic; Submandibular Gland; Terpenes; Thapsigargin