How does regulatory Ca2+ regulate the Na+-Ca2+ exchanger?

Spatial and temporal regulation of intracellular Ca2+ concentrations is a fundamental requirement for life. The mammalian cardiac Na+-Ca2+ exchanger serves as the main mechanism for Ca2+ efflux after heart contraction. Exchange activity is highly regulated by intracellular Ca2+, which binds two regulatory domains (CBD1 and CBD2) and triggers the full activity of the exchanger. We solved the X-ray crystallographic structure of CBD2 in the presence and absence of Ca2+. Together with mutational analysis of the Ca2+ binding sites, this study reveals the crucial role of one of the two bound Ca2+ ions and helps propose hypotheses on the mechanism of regulation of the exchanger.     

Evidence for the involvement of (Cu-ATP)2− in the inhibition of human erythrocyte (Ca2+ + Mg2+-ATPase by copper

The effects of copper on the activity of erythrocyte $\text{(}\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase}$ have been tested on membranes stripped of endogenous calmodulin or recombined with purified calmodulin. The interactions of copper with Ca²⁺, calmodulin and (Mg-ATP)^2? were determined by kinetic studies. The most striking result is the potent competitive inhibition exerted by (Cu-ATP)^2? against (Mg-ATP)^2?$\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 2.8 μ}\text{M}$), while free copper gives no characteristic inhibition. Our results also demonstrate that copper does not compete with calcium either on the enzyme or on calmodulin. The fixation of calmodulin on the enzyme is not altered in the presence of copper as shown by the fact that the dissociation constant remains unaffected. It may be speculated that (Cu-ATP)^2? is the active form of copper, which could plausibly be at the origin of some of the pathological features of erythrocytes observed in conditions associated with excess copper.     

Na+−K+ pump activity and the glucose-stimulated Ca2+-sensitive K+ permeability in the pancreatic B-cell

A rise in the extracellular concentration of glucose from an intermediate to a high value changes the burst pattern of electrical activity of the pancreatic B-cell into a continuous firing, and yet activates the B-cell Ca2+-sensitive K+ permeability. The hypothesis that glucose exerts such effects by inhibiting the Na+, K+-ATPase was investigated. Ouabain (1 mM) mimicked the effect of 16.7 mM glucose in stimulating 86Rb, 45Ca outflow and insulin release from perifused rat pancreatic islets first exposed to 8.3 mM glucose. The stimulation by ouabain of 86Rb outflow was reduced in the absence of extracellular Ca2+ and almost completely abolished in the presence of quinine, and inhibitor of the Ca2+-sensitive K+ permeability. In the presence of ouabain, a rise in the glucose concentration from 8.3 to 16.7 mM failed to stimulate 86Rb outflow. However, the rise in the glucose concentration failed to inhibit 86Rb influx in islet cells, while ouabain dramatically reduced 86Rb influx whether in the presence of 8.3 or 16.7 mM glucose. These findings do not suggest that inhibition of the B-cell Na+, K+-ATPase represents the mechanism by which glucose in high concentration stimulates 86Rb outflow and induces continuous electrical activity in the B-cell.     

Relation between electron exchange and nuclear vibrations in the H 2 + +H 2 + → H 3 + +p exothermic ion-molecular reaction

The effective cross section for the H ₂ ⁺ +H ₂ ⁺ → H ₃ ⁺ +p reaction in the energy range 5.7–11.5 eV is measured by the split beam method. The maximum of the cross section at an energy of ～8 eV is related to the production of the H ₄ ⁺⁺ compound system. The reaction threshold W _thr≈5 eV provides evidence in favor of the classical model of the H ₂ ⁺ ion with the charge fixed on one of the nuclei throughout the collision event.     

Back to the future with the AGP–Ca2+ flux capacitor

Background

Arabinogalactan proteins (AGPs) are ubiquitous in green plants. AGPs comprise a widely varied group of hydroxyproline (Hyp)-rich cell surface glycoproteins (HRGPs). However, the more narrowly defined classical AGPs massively predominate and cover the plasma membrane. Extensive glycosylation by pendant polysaccharides O-linked to numerous Hyp residues like beads of a necklace creates a unique ionic compartment essential to a wide range of physiological processes including germination, cell extension and fertilization. The vital clue to a precise molecular function remained elusive until the recent isolation of small Hyp–arabinogalactan polysaccharide subunits; their structural elucidation by nuclear magentic resonance imaging, molecular simulations and direct experiment identified a 15-residue consensus subunit as a β-1,3-linked galactose trisaccharide with two short branched sidechains each with a single glucuronic acid residue that binds Ca²⁺ when paired with its adjacent sidechain.

Scope

AGPs bind Ca²⁺ (K_d ∼ 6 μm) at the plasma membrane (PM) at pH ∼5·5 but release it when auxin-dependent PM H⁺-ATPase generates a low periplasmic pH that dissociates AGP–Ca²⁺ carboxylates (pk_a ∼3); the consequential large increase in free Ca²⁺ drives entry into the cytosol via Ca²⁺ channels that may be voltage gated. AGPs are thus arguably the primary source of cytosolic oscillatory Ca²⁺ waves. This differs markedly from animals, in which cytosolic Ca²⁺ originates mostly from internal stores such as the sarcoplasmic reticulum. In contrast, we propose that external dynamic Ca²⁺ storage by a periplasmic AGP capacitor co-ordinates plant growth, typically involving exocytosis of AGPs and recycled Ca²⁺, hence an AGP–Ca²⁺ oscillator.

Conclusions

The novel concept of dynamic Ca²⁺ recycling by an AGP–Ca²⁺ oscillator solves the long-standing problem of a molecular-level function for classical AGPs and thus integrates three fields: AGPs, Ca²⁺ signalling and auxin. This accounts for the involvement of AGPs in plant morphogenesis, including tropic and nastic movements.     

Possible role for Ca2+ in the pathophysiology of the prion protein?

Transmissible spongiform encephalopathies, or prion diseases, are lethal neurodegenerative disorders caused by the infectious agent named prion, whose main constituent is an aberrant conformational isoform of the cellular prion protein, PrP(C) . The mechanisms of prion-associated neurodegeneration and the physiologic function of PrP(C) are still unclear, although it is now increasingly acknowledged that PrP(C) plays a role in cell differentiation and survival. PrP(C) thus exhibits dichotomic attributes, as it can switch from a benign function under normal conditions to the triggering of neuronal death during disease. By reviewing data from models of prion infection and PrP-knockout paradigms, here we discuss the possibility that Ca(2+) is the hidden factor behind the multifaceted behavior of PrP(C) . By featuring in almost all processes of cell signaling, Ca(2+) might explain diverse aspects of PrP(C) pathophysiology, including the recently proposed one in which PrP(C) acts as a mediator of synaptic degeneration in Alzheimer's disease.     

Are B-type Ca2+ Channels of Cardiac Myocytes Akin to the Passive Ion Channel in the Plasma Membrane Ca2+ Pump?

The present study demonstrates that B-type Ca²⁺ channels observed in rat ventricular myocytes markedly reacted to agents known to affect the ion-motive plasma membrane Ca²⁺-ATPase (PMCA) pump. Chlorpromazine (CPZ)-activated B-type Ca²⁺ channels were completely blocked by internal application of PMCA pump inhibitors, namely La³⁺ (100 μm), eosin (10 μm) and AIF₃ (100 μm). Calmodulin (50 U/ml), the main endogenous positive regulator of PMCA, was unable to activate but significantly reduced CPZ-activated B-type channel activity. In the same manner, ATP (1 and 4 mm), the main energizing substrate of PMCA, was able to reversibly and significantly reduce this activity in a dose-dependent manner. Interestingly, anti-PMCA antibody 5F10, but not anti-Na/K ATPase antibody (used as a negative control) induced a marked Ba²⁺-conducting channel activity that shared the same characteristics with that of CPZ-activated B-type channels. 5F10-Activated channels were mostly selective towards Ba²⁺, mainly had three observed conductance levels (23, 47 and 85 pS), were observed with a frequency of about 1 out of 5 membrane patches and were completely blocked by 10 μm eosin. These results suggest that B-type Ca²⁺ channels are some form of the PMCA pump. Received: 24 July 2000/Revised: 5 October 2000     

Both Intra- and Extracellular Ca2+ Participate in the Regulation of the Lateral Diffusion of the PDGF-β2 Receptor

When the receptors for platelet-derived growth factor (PDGF) are activatedthey aggregate, become tyrosine-phosphorylated and elicit a cascade ofdown-stream signals, including mobilization of Ca²⁺ from intra- andextracellular stores. Receptor mobility in the plane of the membrane isa prerequisite for receptor aggregation and further signalling. Using humanforeskin fibroblasts (AG 1523) and fluorescence recovery afterphotobleaching (FRAP), we therefore assessed the lateral mobilitycharacteristics of PDGF-2 receptors by their diffusioncoefficient (D), and fraction of mobile receptors (R). This was done oncells stimulated with either normal human serum (NHS) or PDGF underdifferent Ca²⁺-conditions.The results suggest that both intra- and extracellular free Ca²⁺influence the mobility characteristics of the PDGF-2receptor. Interestingly, the extracellular Ca²⁺ seems to imposegeneral restrictions on the mobility of receptors, since R increased whenextracellular Ca²⁺ was quenched with EGTA, whereas intracellularclamping of Ca²⁺ transients with MABTAM (BAPT/AM) primarily affectedD. When both intra- and extracellular Ca²⁺ were quenced, D remainedlow and R high, further supporting the proposition that they achievedistinct effects. Inhibition of tyrosine phosphorylation with Erbstatin,partly inhibited the NHS effects and released PDGF-induced receptorimmobilization. Ratio imaging with Fura-2 displayed that both NHS and PDGFinduced changes in intracellular free [Ca²⁺]. In view of the presentdata it might have important effects on the state of the receptor in themembrane, for instance by regulating its lateral mobility, communicationwith other receptors and signalling functions in the membrane.     

Molecular Basis of the Membrane Interaction of the β2e Subunit of Voltage-Gated Ca2+ Channels

The auxiliary β subunit plays an important role in the regulation of voltage-gated calcium (Ca_V) channels. Recently, it was revealed that β2e associates with the plasma membrane through an electrostatic interaction between N-terminal basic residues and anionic phospholipids. However, a molecular-level understanding of β-subunit membrane recruitment in structural detail has remained elusive. In this study, using a combination of site-directed mutagenesis, liposome-binding assays, and multiscale molecular-dynamics (MD) simulation, we developed a physical model of how the β2e subunit is recruited electrostatically to the plasma membrane. In a fluorescence resonance energy transfer assay with liposomes, binding of the N-terminal peptide (23 residues) to liposome was significantly increased in the presence of phosphatidylserine (PS) and phosphatidylinositol 4,5-bisphosphate (PIP₂). A mutagenesis analysis suggested that two basic residues proximal to Met-1, Lys-2 (K2) and Trp-5 (W5), are more important for membrane binding of the β2e subunit than distal residues from the N-terminus. Our MD simulations revealed that a stretched binding mode of the N-terminus to PS is required for stable membrane attachment through polar and nonpolar interactions. This mode obtained from MD simulations is consistent with experimental results showing that K2A, W5A, and K2A/W5A mutants failed to be targeted to the plasma membrane. We also investigated the effects of a mutated β2e subunit on inactivation kinetics and regulation of Ca_V channels by PIP₂. In experiments with voltage-sensing phosphatase (VSP), a double mutation in the N-terminus of β2e (K2A/W5A) increased the PIP₂ sensitivity of Ca_V2.2 and Ca_V1.3 channels by ∼3-fold compared with wild-type β2e subunit. Together, our results suggest that membrane targeting of the β2e subunit is initiated from the nonspecific electrostatic insertion of N-terminal K2 and W5 residues into the membrane. The PS-β2e interaction observed here provides a molecular insight into general principles for protein binding to the plasma membrane, as well as the regulatory roles of phospholipids in transporters and ion channels.     

Tetracyclines modulate cytosolic Ca2+ responses in the osteoclast associated with “Ca2+ receptor” activation

We report the effects of tetracycline analogues on cytosolic Ca²⁺ transients resulting from application of ionic nickel (Ni²⁺), a potent surrogate agonist of the osteoclast Ca²⁺ receptor. Preincubation with minocycline (1 mg/l) or a chemically modified tetracycline, 4-dedimethyl-aminotetracycline (CMT-1) (1 or 10 mg/l), resulted in a significant attenuation of the magnitude of the cytosolic [Ca²⁺] response to an application of 5 mM-[Ni²⁺]. Preincubation with doxycycline (1 or 10 mg/l) failed to produce similar results. In addition, application of minocycline alone (0.1–100 mg/l) resulted in a 3.5-fold elevation of cytosolic [Ca²⁺]. The results suggest a novel action of tetracyclines on the osteoclast Ca²⁺ receptor.     

Ca2+-antagonists inhibit the N-methyltransferase‐dependent synthesis of phosphatidylcholine in the heart

Evidence indicates that, in addition to the Ltype Ca²⁺ channel blockade, Ca²⁺antagonists target other functions including the Ca²⁺pumps. This study was conducted to test the possibility that the reported inhibition of heart sarcolemmal (SL) and sarcoplasmic reticular (SR) Ca²⁺pumps by verapamil and diltiazem could be due to druginduced depression of phosphatidylethanolamine (PE) Nmethylation which modulates these Ca²⁺transport systems. Three catalytic sites individually responsible for the synthesis of PE monomethyl (site I), dimethyl (site II) and trimethyl (phosphatidylcholine (PC), site III) derivates were examined in SL and SR membranes by employing different concentrations of SadenosylLmethionine (AdoMet). Total methyl group incorporation into SL PE, in vitro, was significantly depressed by 10^–6–10^–3 M verapamil or diltiazem at site III. The catalytic activity of site I was inhibited by 10^–3 M verapamil only, whereas the site II activity was not affected by these drugs. The inhibition induced by verapamil or diltiazem (10^–5 M) was associated with a depression of the V_max value without any change in the apparent affinity for AdoMet. Both drugs decreased the SR as well as mitochondrial PE Nmethylation at site III. A selective depression of site III activity was also observed in SL isolated from hearts of rats treated with verapamil in vivo. Furthermore, administration of [³H-methyl]methionine following the treatment of animals with verapamil, reduced the synthesis of PC by Nmethyltransferase. Verapamil also depressed the N-methylation-dependent positive inotropic effect induced by methionine in the isolated Langendorff heart. Both agents depressed the SL Ca²⁺pump and although diltiazem also inhibited the SR Ca²⁺pump, verapamil exerted a stimulatory effect. In addition, verapamil decreased SR Ca²⁺-release. These results suggest that verapamil and diltiazem alter the cardiac PE Nmethyltransferase system. This action is apparently additional to the drugs' effect on Ltype Ca²⁺ channels and may serve as a biochemical mechanism for the drugs' inhibition of the cardiac Ca²⁺pumps and altered cardiac function.     

Measuring the Influence of the BKCa β1 Subunit on Ca2+ Binding to the BKCa Channel

The large-conductance Ca²⁺-activated potassium (BK_Ca) channel of smooth muscle is unusually sensitive to Ca²⁺ as compared with the BK_Ca channels of brain and skeletal muscle. This is due to the tissue-specific expression of the BK_Ca auxiliary subunit β1, whose presence dramatically increases both the potency and efficacy of Ca²⁺ in promoting channel opening. β1 contains no Ca²⁺ binding sites of its own, and thus the mechanism by which it increases the BK_Ca channel''s Ca²⁺ sensitivity has been of some interest. Previously, we demonstrated that β1 stabilizes voltage sensor activation, such that activation occurs at more negative voltages with β1 present. This decreases the work that Ca²⁺ must do to open the channel and thereby increases the channel''s apparent Ca²⁺ affinity without altering the real affinities of the channel''s Ca²⁺ binding sites. To explain the full effect of β1 on the channel''s Ca²⁺ sensitivity, however, we also proposed that there must be effects of β1 on Ca²⁺ binding. Here, to test this hypothesis, we have used high-resolution Ca²⁺ dose–response curves together with binding site–specific mutations to measure the effects of β1 on Ca²⁺ binding. We find that coexpression of β1 alters Ca²⁺ binding at both of the BK_Ca channel''s two types of high-affinity Ca²⁺ binding sites, primarily increasing the affinity of the RCK1 sites when the channel is open and decreasing the affinity of the Ca²⁺ bowl sites when the channel is closed. Both of these modifications increase the difference in affinity between open and closed, such that Ca²⁺ binding at either site has a larger effect on channel opening when β1 is present.     

N-terminal Isoforms of the Large-conductance Ca2+-activated K+ Channel Are Differentially Modulated by the Auxiliary β1-Subunit

The large-conductance Ca²⁺-activated K⁺ (BK_Ca) channel is essential for maintaining the membrane in a hyperpolarized state, thereby regulating neuronal excitability, smooth muscle contraction, and secretion. The BK_Ca α-subunit has three predicted initiation codons that generate proteins with N-terminal ends starting with the amino acid sequences MANG, MSSN, or MDAL. Because the N-terminal region and first transmembrane domain of the α-subunit are required for modulation by auxiliary β1-subunits, we examined whether β1 differentially modulates the N-terminal BK_Ca α-subunit isoforms. In the absence of β1, all isoforms had similar single-channel conductances and voltage-dependent activation. However, whereas β1 did not modulate the voltage-activation curve of MSSN, β1 induced a significant leftward shift of the voltage activation curves of both the MDAL and MANG isoforms. These shifts, of which the MDAL was larger, occurred at both 10 μm and 100 μm Ca²⁺. The β1-subunit increased the open dwell times of all three isoforms and decreased the closed dwell times of MANG and MDAL but increased the closed dwell times of MSSN. The distinct modulation of voltage activation by the β1-subunit may be due to the differential effect of β1 on burst duration and interburst intervals observed among these isoforms. Additionally, we observed that the related β2-subunit induced comparable leftward shifts in the voltage-activation curves of all three isoforms, indicating that the differential modulation of these isoforms was specific to β1. These findings suggest that the relative expression of the N-terminal isoforms can fine-tune BK_Ca channel activity in cells, highlighting a novel mechanism of BK_Ca channel regulation.     

Theoretical study of the hydroxylation of phenolates by the Cu2O2(N,N′-dimethylethylenediamine)2 2+ complex

Tyrosinase catalyzes the ortho hydroxylation of monophenols and the subsequent oxidation of the diphenolic products to the resulting quinones. In efforts to create biomimetic copper complexes that can oxidize C–H bonds, Stack and coworkers recently reported a synthetic μ-η²:η²-peroxodicopper(II)(DBED)₂ complex (DBED is N,N′-di-tert-butylethylenediamine), which rapidly hydroxylates phenolates. A reactive intermediate consistent with a bis-μ-oxo-dicopper(III)-phenolate complex, with the O–O bond fully cleaved, is observed experimentally. Overall, the evidence for sequential O–O bond cleavage and C–O bond formation in this synthetic complex suggests an alternative mechanism to the concerted or late-stage O–O bond scission generally accepted for the phenol hydroxylation reaction performed by tyrosinase. In this work, the reaction mechanism of this peroxodicopper(II) complex was studied with hybrid density functional methods by replacing DBED in the μ-η²:η²-peroxodicopper(II)(DBED)₂ complex by N,N′-dimethylethylenediamine ligands to reduce the computational costs. The reaction mechanism obtained is compared with the existing proposals for the catalytic ortho hydroxylation of monophenol and the subsequent oxidation of the diphenolic product to the resulting quinone with the aim of gaining some understanding about the copper-promoted oxidation processes mediated by 2:1 Cu(I)O₂-derived species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Ca2+ binding to synaptotagmin: how many Ca2+ ions bind to the tip of a C2-domain?

C2-domains are widespread protein modules with diverse Ca2+-regulatory functions. Although multiple Ca2+ ions are known to bind at the tip of several C2-domains, the exact number of Ca2+-binding sites and their functional relevance are unknown. The first C2-domain of synaptotagmin I is believed to play a key role in neurotransmitter release via its Ca2+-dependent interactions with syntaxin and phospholipids. We have studied the Ca2+-binding mode of this C2-domain as a prototypical C2-domain using NMR spectroscopy and site-directed mutagenesis. The C2-domain is an elliptical module composed of a beta-sandwich with a long axis of 50 A. Our results reveal that the C2-domain binds three Ca2+ ions in a tight cluster spanning only 6 A at the tip of the module. The Ca2+-binding region is formed by two loops whose conformation is stabilized by Ca2+ binding. Binding involves one serine and five aspartate residues that are conserved in numerous C2-domains. All three Ca2+ ions are required for the interactions of the C2-domain with syntaxin and phospholipids. These results support an electrostatic switch model for C2-domain function whereby the beta-sheets of the domain provide a fixed scaffold for the Ca2+-binding loops, and whereby interactions with target molecules are triggered by a Ca2+-induced switch in electrostatic potential.