Synthetic peptide K+ carrier with Ca2+ inhibition

Based on a proposed solution conformation of the Ca²⁺ ion complex of the repeat hexapeptide of elastin, l-Val-l-Ala-l-Pro-Gly-l-Val-Gly, it is possible to modify the molecule making it more lipophilic for lipid bilayer permeation while retaining its complexation features. Therefore the two peptides, For-MeVal-Ala-Pro-Sar-Pro-Sar-OMe and For-MeVal-Ala-Pro-Sar-Pro-Sar-OH, were synthesized and evaluated for lipid bilayer activity and cation binding (For, N-formyl; Me, N-methyl; Sar, N-methyl glycine). Both peptides bound Ca²⁺ preferentially but did not exhibit the properties of a Ca²⁺ carrier. They were however active as K⁺ carriers although K⁺ ion titration curves showed a much lower affinity for K⁺ than for Ca²⁺. The addition of Ca²⁺ or Mg²⁺ to the bilayer system inhibited the peptide K⁺ carrier activity. Three possible explanations of this interesting Ca²⁺ inhibition of carrier activity are irreversible complexation of Ca²⁺, mixed ligand complex formation involving Ca²⁺, lipid and peptide, and impermeability of the lipid layer when peptide is complexed with a divalent cation.     

In situ Ca2+ titration in the fluorometric study of intracellular Ca2+ binding

Imaging with Ca²⁺-sensitive fluorescent dye has provided a wealth of insight into the dynamics of cellular Ca²⁺ signaling. The spatiotemporal evolution of intracellular free Ca²⁺ observed in imaging experiments is shaped by binding and unbinding to cytoplasmic Ca²⁺ buffers, as well as the fluorescent indicator used for imaging. These factors must be taken into account in the interpretation of Ca²⁺ imaging data, and can be exploited to investigate endogenous Ca²⁺ buffer properties. Here we extended the use of Ca²⁺ fluorometry in the characterization of Ca²⁺ binding molecules within cells, building on a method of titration of intracellular Ca²⁺ binding sites in situ with measured amounts of Ca²⁺ entering through voltage-gated Ca²⁺ channels. We developed a systematic procedure for fitting fluorescence data acquired during a series of voltage steps to models with multiple Ca²⁺ binding sites. The method was tested on simulated data, and then applied to 2-photon fluorescence imaging data from rat posterior pituitary nerve terminals patch clamp-loaded with the Ca²⁺ indicator fluo-8. Focusing on data sets well described by a single endogenous Ca²⁺ buffer and dye, this method yielded estimates of the endogenous buffer concentration and K_d, the dye K_d, and the fraction of Ca²⁺ inaccessible cellular volume. The in situ K_d of fluo-8 thus obtained was indistinguishable from that measured in vitro. This method of calibrating Ca²⁺-sensitive fluorescent dyes in situ has significant advantages over previous methods. Our analysis of Ca²⁺ titration fluorometric data makes more effective use of the experimental data, and provides a rigorous treatment of multivariate errors and multiple Ca²⁺ binding species. This method offers a versatile approach to the study of endogenous Ca²⁺ binding molecules in their physiological milieu.     

The KN-93 Molecule Inhibits Calcium/Calmodulin-Dependent Protein Kinase II (CaMKII) Activity by Binding to Ca2+/CaM

Calcium/calmodulin-dependent protein kinase II (CaMKII) is a multifunctional serine/threonine protein kinase that transmits calcium signals in various cellular processes. CaMKII is activated by calcium-bound calmodulin (Ca²⁺/CaM) through a direct binding mechanism involving a regulatory C-terminal α-helix in CaMKII. The Ca²⁺/CaM binding triggers transphosphorylation of critical threonine residues proximal to the CaM-binding site leading to the autoactivated state of CaMKII. The demonstration of its critical roles in pathophysiological processes has elevated CaMKII to a key target in the management of numerous diseases. The molecule KN-93 is the most widely used inhibitor for studying the cellular and in vivo functions of CaMKII. It is widely believed that KN-93 binds directly to CaMKII, thus preventing kinase activation by competing with Ca²⁺/CaM. Herein, we employed surface plasmon resonance, NMR, and isothermal titration calorimetry to characterize this presumed interaction. Our results revealed that KN-93 binds directly to Ca²⁺/CaM and not to CaMKII. This binding would disrupt the ability of Ca²⁺/CaM to interact with CaMKII, effectively inhibiting CaMKII activation. Our findings also indicated that KN-93 can specifically compete with a CaMKIIδ-derived peptide for binding to Ca²⁺/CaM. As indicated by the surface plasmon resonance and isothermal titration calorimetry data, apparently at least two KN-93 molecules can bind to Ca²⁺/CaM. Our findings provide new insight into how in vitro and in vivo data obtained with KN-93 should be interpreted. They further suggest that other Ca²⁺/CaM-dependent, non-CaMKII activities should be considered in KN-93–based mechanism-of-action studies and drug discovery efforts.     

A charge‐sensitive loop in the FKBP38 catalytic domain modulates Bcl‐2 binding

The Bcl‐2 inhibitor FKBP38 is regulated by the Ca²⁺‐sensor calmodulin (CaM). Here we show a hitherto unknown low‐affinity cation‐binding site in the FKBP domain of FKBP38, which may afford an additional level of regulation based on electrostatic interactions. Fluorescence titration experiments indicate that in particular the physiologically relevant Ca²⁺ ion binds to this site. NMR‐based chemical shift perturbation data locate this cation‐interaction site within the β5–α1 loop (Leu90–Ile96) of the FKBP domain, which contains the acidic Asp92 and Asp94 side‐chains. Binding constants were subsequently determined for K⁺, Mg²⁺, Ca²⁺, and La³⁺, indicating that the net charge and the radius of the ion influences the binding interaction. X‐ray diffraction data furthermore show that the conformation of the β5–α1 loop is influenced by the presence of a positively charged guanidinium group belonging to a neighboring FKBP38 molecule in the crystal lattice. The position of the cation‐binding site has been further elucidated based on pseudocontact shift data obtained by NMR via titration with Tb³⁺. Elimination of the Ca²⁺‐binding capacity by substitution of the respective aspartate residues in a D92N/D94N double‐substituted variant reduces the Bcl‐2 affinity of the FKBP38^35–153/CaM complex to the same degree as the presence of Ca²⁺ in the wild‐type protein. Hence, this charge‐sensitive site in the FKBP domain participates in the regulation of FKBP38 function by enabling electrostatic interactions with ligand proteins and/or salt ions such as Ca²⁺. Copyright © 2010 John Wiley & Sons, Ltd.     

Conformational Studies on Calcium Binding By tBoc-Leu-Pro-Tyr-Ala-NHCH3, a Tyrosine Kinase Substrate,in a Nonpolar Solvent

Abstract

With a view to understanding the structural requirement for tyrosine phosphorylation, we have examined the free and Ca²⁺-bound conformations of the synthetic peptide tBoc-Leu-Pro-Tyr-Ala-NHCH₃, a substrate for a protein tyrosine kinase, using circular dichroism (CD), ¹H and ¹³C nuclear magnetic resonance (NMR) and molecular modeling methods. CD spectrum of the free peptide in water showed a random coil structure, while the spectrum in acetonitrile was indicative of a folded structure containing a type III β-turn. Dihedral angle data derived from J_NH-CH coupling constants, as well as two-dimensional ¹H-COSY and NOESY spectral analyses, showed that the peptide adopts a conformation close to the 3_10- helix. Ca²⁺ binding by the peptide, as monitored by CD spectral changes, was quite weak in water. However, substantial CD spectral changes were observed in the peptide on addition of Ca²⁺ in acetonitrile suggestive of major conformational alterations due to Ca²⁺ binding. Analysis of the binding isotherms at 25°C obtained from CD data in acetonitrile indicated a 2:1 peptide:Ca²⁺ (“sandwich”) complex to be the dominant species with a K_d of about 30μM. A. 1:1 complex was also present and became significant at Ca²⁺:peptide ratios above 1. By comparison, the peptide formed a predominantly 1:1 complex with Mg²⁺ with a K_d of about 40μM. ¹³C-NMR data showed that a mixture of cis and trans conformers (arising from rotation around the Leu-Pro bond) in the free peptide changes over to the all-trans form on coordination of the peptide carbonyl groups to the Ca²⁺ ion. ¹H-NOESY data of the Ca²⁺ complex revealed several interactions involving the sidechains of two peptide molecules in the sandwich. Molecular modeling and energy minimization with and without the input of NOESY-derived distance constraints showed the sandwich complex to be an energetically very favourable conformation. Besides its relevance in terms of the possible involvement of divalent cations in substrate-tyrosine kinase interaction, the conformational characterization of tBoc-Leu-Pro-Tyr-Ala-NHCH₃ and its Ca²⁺ complex should help understand the conformational determinants for Ca²⁺-binding by linear peptides.     

Specificity of ligand binding to transport sites: Ca2+ binding to the Ca2+ transport ATPase and its dependence on H+ and Mg2+

Ligand binding to transport sites constitutes the initial step in the catalytic cycle of transport ATPases. Here, we consider the well characterized Ca²⁺ ATPase of sarcoplasmic reticulum (SERCA) and describe a series of Ca²⁺ binding isotherms obtained by equilibrium measurements in the presence of various H⁺ and Mg²⁺ concentrations. We subject the isotherms to statistical mechanics analysis, using a model based on a minimal number of mechanistic steps. The analysis allows satisfactory fits and yields information on occupancy of the specific Ca²⁺ sites under various conditions. It also provides a fundamental method for analysis of binding specificity to transport sites under equilibrium conditions that lead to tightly coupled catalytic activation.     

Membrane Modulates Affinity for Calcium Ion to Create an Apparent Cooperative Binding Response by Annexin a5

Isothermal titration calorimetry was used to characterize the binding of calcium ion (Ca²⁺) and phospholipid to the peripheral membrane-binding protein annexin a5. The phospholipid was a binary mixture of a neutral and an acidic phospholipid, specifically phosphatidylcholine and phosphatidylserine in the form of large unilamellar vesicles. To stringently define the mode of binding, a global fit of data collected in the presence and absence of membrane concentrations exceeding protein saturation was performed. A partition function defined the contribution of all heat-evolving or heat-absorbing binding states. We find that annexin a5 binds Ca²⁺ in solution according to a simple independent-site model (solution-state affinity). In the presence of phosphatidylserine-containing liposomes, binding of Ca²⁺ differentiates into two classes of sites, both of which have higher affinity compared with the solution-state affinity. As in the solution-state scenario, the sites within each class were described with an independent-site model. Transitioning from a solution state with lower Ca²⁺ affinity to a membrane-associated, higher Ca²⁺ affinity state, results in cooperative binding. We discuss how weak membrane association of annexin a5 prior to Ca²⁺ influx is the basis for the cooperative response of annexin a5 toward Ca²⁺, and the role of membrane organization in this response.     

Identification of Voltage Operated Calcium Channels by Binding Studies: Differentiation of Subclasses of Calcium Antagonist Drugs with 3H-Nimodipine Radioligand Binding

Abstract

³H-Nimodipine (³H -NIM) is a high affinity radioligand suitable to study Ca²⁺ -channels in a variety of tissues. The binding is saturable, reversible, and stereospecific in purified bovine heart and partially purified guinea-pig brain membranes. In the latter a Bmax of 600fmol/mg protein, dissociation constants (K_D) of 0.4-0.8nM and a Hill slope of 1.0 are found. At 37^C the optimal pH in 50mM TRIS-HCl buffer is 7.1-7.4. The calcium channel is a metalloprotein, and the divalent cation which is essential for the binding of ³ H -NIM can be removed by EDTA (EC₅₀ 20μM); the nimodipine binding site of the channel may then be reconstituted by divalent cations with Mn²⁺ > Ca²⁺>Mg²⁺>Sr²⁺. Ca²⁺-antagonist drugs can be divided into three main classes based on their interaction with the ³H -NIM binding site: Class I has one site law of mass action-displacement isotherms with ³H -NIM, Class II exhibits complex biphasic inhibition profiles and Class III drugs increase the affinity of 1,4 dihydropyridines for the Ca²⁺ -channel. Diltiazem is a Class III Ca²⁺ -antagonist. Our in vitro studies lead us to conclude that the Ca²⁺ -channel contains multiple regulatory sites at which drugs can act.     

Crystal Structure of CBD2 from the Drosophila Na/Ca Exchanger: Diversity of Ca Regulation and Its Alternative Splicing Modification

Na⁺/Ca²⁺ exchangers (NCXs) promote the extrusion of intracellular Ca²⁺ to terminate numerous Ca²⁺-mediated signaling processes. Ca²⁺ interaction at two Ca²⁺ binding domains (CBDs; CBD1 and CBD2) is important for tight regulation of the exchange activity. Diverse Ca²⁺ regulatory properties have been reported with several NCX isoforms; whether the regulatory diversity of NCXs is related to structural differences of the pair of CBDs is presently unknown. Here, we reported the crystal structure of CBD2 from the Drosophila melanogaster exchanger CALX1.1. We show that the CALX1.1-CBD2 is an immunoglobulin-like structure, similar to mammalian NCX1-CBD2, but the predicted Ca²⁺ interaction region of CALX1.1-CBD2 is arranged in a manner that precludes Ca²⁺ binding. The carboxylate residues that coordinate two Ca²⁺ in the NCX1-CBD1 structure are neutralized by two Lys residues in CALX1.1-CBD2. This structural observation was further confirmed by isothermal titration calorimetry. The CALX1.1-CBD2 structure also clearly shows the alternative splicing region forming two adjacent helices perpendicular to CBD2. Our results provide structural evidence that the diversity of Ca²⁺ regulatory properties of NCX proteins can be achieved by (1) local structure rearrangement of Ca²⁺ binding site to change Ca²⁺ binding properties of CBD2 and (2) alternative splicing variation altering the protein domain-domain conformation to modulate the Ca²⁺ regulatory behavior.     

Measurement of the binding parameters of annexin derivative-erythrocyte membrane interactions

Erythrocyte ghosts prepared from fresh blood expressed phosphatidylserine (PS) on the membrane surfaces in a rather stable fashion. The binding of fluorescein-5-isothiocyanate (FITC)-labeled annexin V (ANV) derivatives to these membranes was studied by titration with proteins and with calcium. Whereas the preaddition of ethylenediaminetetraacetic acid (EDTA) to reaction mixtures totally prevented membrane binding, Ca²⁺-dependent binding was only partially reversed by EDTA treatment, consistent with an initial Ca²⁺-dependent binding that became partially Ca²⁺ independent. Data derived from saturation titration with ANV derivatives poorly fit the simple protein-membrane equilibrium binding equation and showed negative cooperativity of binding with increasing membrane occupancy. In contrast, calcium titration at low binding site occupancy resulted in excellent fit into the protein-Ca²⁺-membrane equilibrium binding equation. Calcium titrations of FITC-labeled ANV and ANV-6L15 (a novel ANV-Kunitz protease inhibitor fusion protein) yielded a Hill coefficient of approximately 4 in both cases. The apparent dissociation constant for ANV-6L15 was approximately 4-fold lower than that of ANV at 1.2-2.5 mM Ca²⁺. We propose that ANV-6L15 may provide improved detection of PS exposed on the membrane surfaces of pathological cells in vitro and in vivo.     

The interaction of Ca2+ with human factor IX

The binding isotherms of Ca²⁺ and Sr²⁺ to human blood coagulation Factor IX have been obtained at 25 °C and pH 7.4. In the case of both cations, a Scatchard plot of the data reveals that a single class of binding sites exist. For Ca²⁺, a total of 16.0 ± 1.0 sites, of K_D 7.3 ± 0.2 × 10^?4m, are present on human Factor IX. Similar analysis of the Sr²⁺ data indicates that Factor IX contains 11.0 ± 1.0 binding sites, with a K_D of 1.9 ± 0.1 × 10^?3m. Both Sr²⁺ and Mn²⁺ effectively displace Ca²⁺ from human Factor IX; whereas Mg²⁺ is considerably less potent in this regard. Conversely, Ca²⁺ is capable of nearly complete displacement of Sr²⁺ from its binding sites on human Factor IX. The activation of human Factor IX, by human Factor XIa, shows a complex dependence on the Ca²⁺ concentration. Sr²⁺ can substitute for Ca²⁺ in this activation process. Mn²⁺ cannot, in itself, substitute for Ca²⁺ in activation of Factor IX, but does significantly enhance the activation of Factor IX by Factor XIa at suboptimal levels of Ca²⁺. The rate of activation of human Factor IX by the coagulant protein of Russell's viper venom also shows a dependence on the presence of divalent cations. Here, however, a rigid specificity is not noted, since Ca²⁺, Sr²⁺, and Mn²⁺ all allow activation to proceed equally well.     

Predicting Ca2+‐binding sites using refined carbon clusters

Identifying Ca²⁺‐binding sites in proteins is the first step toward understanding the molecular basis of diseases related to Ca²⁺‐binding proteins. Currently, these sites are identified in structures either through X‐ray crystallography or NMR analysis. However, Ca²⁺‐binding sites are not always visible in X‐ray structures due to flexibility in the binding region or low occupancy in a Ca²⁺‐binding site. Similarly, both Ca²⁺ and its ligand oxygens are not directly observed in NMR structures. To improve our ability to predict Ca²⁺‐binding sites in both X‐ray and NMR structures, we report a new graph theory algorithm (MUG^C) to predict Ca²⁺‐binding sites. Using carbon atoms covalently bonded to the chelating oxygen atoms, and without explicit reference to side‐chain oxygen ligand co‐ordinates, MUG^C is able to achieve 94% sensitivity with 76% selectivity on a dataset of X‐ray structures composed of 43 Ca²⁺‐binding proteins. Additionally, prediction of Ca²⁺‐binding sites in NMR structures was obtained by MUG^C using a different set of parameters, which were determined by the analysis of both Ca²⁺‐constrained and unconstrained Ca²⁺‐loaded structures derived from NMR data. MUG^C identified 20 of 21 Ca²⁺‐binding sites in NMR structures inferred without the use of Ca²⁺ constraints. MUG^C predictions are also highly selective for Ca²⁺‐binding sites as analyses of binding sites for Mg²⁺, Zn²⁺, and Pb²⁺ were not identified as Ca²⁺‐binding sites. These results indicate that the geometric arrangement of the second‐shell carbon cluster is sufficient not only for accurate identification of Ca²⁺‐binding sites in NMR and X‐ray structures but also for selective differentiation between Ca²⁺ and other relevant divalent cations. © Proteins 2012. © 2012 Wiley Periodicals, Inc.     

The binding of phosphorylase kinase to immobilized calmodulin

The binding of phosphorylase kinase to calmodulin-Sepharose 4B was studied by column and batch methods. It was found that the Ca²⁺ dependence of the interaction strongly depended strongly depended on the degree of substitution of agarose with calmodulin. Equilibrium adsorption isotherms (i.e., bulk ligand binding functions and lattice site binding functions) of phosphorylase kinase were measured on calmodulin-Sepharose. Sigmoidal bulk ligand binding functions (bulk adsorption coefficients: 1.5–5.8) were found which indicate intermolecular attraction during binding. Hyperbolic lattice site binding functions (lattice adsorption coefficients: 1.0) were obtained thus excluding the existence of a critical surface concentration of immobilized calmodulin and indicating single independent binding sites on the gel surface and on phosphorylase kinase. These findings were combined to optimize the adsorption of phosphorylase kinase on calmodulin-Sepharose, for purification procedures at low Ca²⁺ concentrations (5–10 μM ) minimizing proteolysis by calpains. With this novel method phosphorylase kinase from rabbit and frog skeletal muscle could be purified ca 100- and 200-fold, respectively, in two steps.     

Mechanism of stimulation of Ca2+ plus Mg2+ -dependent phospodiesterase from rat cerebral cortex by the modulator protein and Ca2+

The activity of phosphodiesterase (“Ca²⁺ plus Mg²⁺-dependent” phosphodiesterase) of a preparation from brain was found to depend on the presence of both Ca²⁺ and a protein factor called modulator. It was shown by gel filtration that the active enzyme-modulator complex (MW, about 200,000) was formed from the modulator (MW, 28,000) and an inactive enzyme (MW, about 150,000) in the presence of Ca²⁺. When EGTA was added, this active enzyme-modulator complex dissociated into inactive enzyme and modulator. These results, together with the finding of Teo and Wang that Ca²⁺ binds to the modulator, could explain the stimulatory effect of Ca²⁺ on this enzyme as follows: The “Ca²⁺ plus Mg²⁺-dependent” phosphodiesterase may exist as the inactive free form in equilibrium with the active enzymemodulator (Ca²⁺) complex, and Ca²⁺, through binding to the modulator, may shift the equilibrium towards formation of the active enzyme-modulator (Ca²⁺) complex, thereby increasing the activity of the mixture. On decreasing the concentration of Ca²⁺, the process is reversible.     

Ca2+-dependent Structural Changes in the B-cell Receptor CD23 Increase Its Affinity for Human Immunoglobulin E

Immunoglobulin E (IgE) antibodies play a fundamental role in allergic disease and are a target for therapeutic intervention. IgE functions principally through two receptors, FcϵRI and CD23 (FcϵRII). Minute amounts of allergen trigger mast cell or basophil degranulation by cross-linking IgE-bound FcϵRI, leading to an inflammatory response. The interaction between IgE and CD23 on B-cells regulates IgE synthesis. CD23 is unique among Ig receptors in that it belongs to the C-type (calcium-dependent) lectin-like superfamily. Although the interaction of CD23 with IgE is carbohydrate-independent, calcium has been reported to increase the affinity for IgE, but the structural basis for this activity has previously been unknown. We have determined the crystal structures of the human lectin-like head domain of CD23 in its Ca²⁺-free and Ca²⁺-bound forms, as well as the crystal structure of the Ca²⁺-bound head domain of CD23 in complex with a subfragment of IgE-Fc consisting of the dimer of Cϵ3 and Cϵ4 domains (Fcϵ3-4). Together with site-directed mutagenesis, the crystal structures of four Ca²⁺ ligand mutants, isothermal titration calorimetry, surface plasmon resonance, and stopped-flow analysis, we demonstrate that Ca²⁺ binds at the principal and evolutionarily conserved binding site in CD23. Ca²⁺ binding drives Pro-250, at the base of an IgE-binding loop (loop 4), from the trans to the cis configuration with a concomitant conformational change and ordering of residues in the loop. These Ca²⁺-induced structural changes in CD23 lead to additional interactions with IgE, a more entropically favorable interaction, and a 30-fold increase in affinity of a single head domain of CD23 for IgE. Taken together, these results suggest that binding of Ca²⁺ brings an extra degree of modulation to CD23 function.     

Interaction of a tyrosine kinase inhibitor,vandetanib with human serum albumin as studied by fluorescence quenching and molecular docking

Interaction of a tyrosine kinase inhibitor, vandetanib (VDB), with the major transport protein in the human blood circulation, human serum albumin (HSA), was investigated using fluorescence spectroscopy, circular dichroism (CD) spectroscopy, and molecular docking analysis. The binding constant of the VDB–HSA system, as determined by fluorescence quenching titration method was found in the range, 8.92–6.89?×?10^3?M^?1 at three different temperatures, suggesting moderate binding affinity. Furthermore, decrease in the binding constant with increasing temperature revealed involvement of static quenching mechanism, thus affirming the formation of the VDB–HSA complex. Thermodynamic analysis of the binding reaction between VDB and HSA yielded positive ΔS (52.76 J?mol^?1 K^?1) and negative ΔH (?6.57?kJ?mol^?1) values, which suggested involvement of hydrophobic interactions and hydrogen bonding in stabilizing the VDB–HSA complex. Far-UV and near-UV CD spectral results suggested alterations in both secondary and tertiary structures of HSA upon VDB-binding. Three-dimensional fluorescence spectral results also showed significant microenvironmental changes around the Trp residue of HSA consequent to the complex formation. Use of site-specific marker ligands, such as phenylbutazone (site I marker) and diazepam (site II marker) in competitive ligand displacement experiments indicated location of the VDB binding site on HSA as Sudlow’s site I (subdomain IIA), which was further established by molecular docking results. Presence of some common metal ions, such as Ca²⁺, Zn²⁺, Cu²⁺, Ba²⁺, Mg²⁺, and Mn²⁺ in the reaction mixture produced smaller but significant alterations in the binding affinity of VDB to HSA.     

Ruthenium red as a resonance Raman probe of Ca2+ binding sites in biological materials.

We have observed substantial changes in the resonance Raman spectrum of ruthenium red when it is added to calcium ion binding molecules and organelles, including proteins, phospholipids, chelating agents, and intact mitochondria. The addition of Ca²⁺ ions can reverse these observed spectral changes. In the case of cytochrome c, ruthenium red binding varies with oxidation state in a manner parallel to that for Ca²⁺ binding. The resonance Raman spectrum of a ruthenium red-phospholipid complex shows differences from that of a ruthenium red-protein complex, enabling us to distinguish between binding to these different classes of molecules. Our studies suggest that the primary constituent of the low-affinity Ca²⁺ binding sites in mitochondria is cardiolipin.     

Kinetics on Demand Is a Simple Mathematical Solution that Fits Recorded Caffeine-Induced Luminal SR Ca2+ Changes in Smooth Muscle Cells

The process of Ca²⁺ release from sarcoplasmic reticulum (SR) comprises 4 phases in smooth muscle cells. Phase 1 is characterized by a large increase of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) with a minimal reduction of the free luminal SR [Ca²⁺] ([Ca²⁺]_FSR). Importantly, active SR Ca²⁺ ATPases (SERCA pumps) are necessary for phase 1 to occur. This situation cannot be explained by the standard kinetics that involves a fixed amount of luminal Ca²⁺ binding sites. A new mathematical model was developed that assumes an increasing SR Ca²⁺ buffering capacity in response to an increase of the luminal SR [Ca²⁺] that is called Kinetics-on-Demand (KonD) model. This approach can explain both phase 1 and the refractory period associated with a recovered [Ca²⁺]_FSR. Additionally, our data suggest that active SERCA pumps are a requisite for KonD to be functional; otherwise luminal SR Ca²⁺ binding proteins switch to standard kinetics. The importance of KonD Ca²⁺ binding properties is twofold: a more efficient Ca²⁺ release process and that [Ca²⁺]_FSR and Ca²⁺-bound to SR proteins ([Ca²⁺]_BSR) can be regulated separately allowing for Ca²⁺ release to occur (provided by Ca²⁺-bound to luminal Ca²⁺ binding proteins) without an initial reduction of the [Ca²⁺]_FSR.     

Potential cation and H+ binding sites in acid sensing ion channel-1

Acid sensing ion channels (ASICs) are cation-selective membrane channels activated by H⁺ binding upon decrease in extracellular pH. It is known that Ca²⁺ plays an important modulatory role in ASIC gating, competing with the ligand (H⁺) for its binding site(s). However, the H⁺ or Ca²⁺ binding sites involved in gating and the gating mechanism are not fully known. We carried out a computational study to investigate potential cation and H⁺ binding sites for ASIC1 via all-atom molecular dynamics simulations on five systems. The systems were designed to test the candidacy of some acid sensing residues proposed from experiment and to determine yet unknown ligand binding sites. The ion binding patterns reveal sites of cation (Na⁺ and Ca²⁺) localization where they may compete with protons and influence channel gating. The highest incidence of Ca²⁺ and Na⁺ binding is observed at a highly acidic pocket on the protein surface. Also, Na⁺ ions fill in an inner chamber that contains a ring of acidic residues and that is near the channel entrance; this site could possibly be a temporary reservoir involved in ion permeation. Some acidic residues were observed to orient and move significantly close together to bind Ca²⁺, indicating the structural consequences of Ca²⁺ release from these sites. Local structural changes in the protein due to cation binding or ligand binding (protonation) are examined at the binding sites and discussed. This study provides structural and dynamic details to test hypotheses for the role of Ca²⁺ and Na⁺ ions in the channel gating mechanism.