Conformational changes occurring in N-ras p21 in response to binding of guanine nucleotide and metal ions probed by proteolysis performed under controlled conditions

Variations in susceptibility to proteolysis by trypsin and chymotrypsin have been used as indicators of conformational changes taking place in N-ras p21 in response to ligand binding. It has been observed that changes occur in undenatured protein, rendering it more resistant to degradation, in the presence of divalent cations such as Mg2+ and Ca2+ (suggesting direct binding of metals to the polypeptide) and even more markedly in the presence of GDP and/or Mg2+ GDP. Monovalent cations (Na+ or K+) cannot substitute for Mg2+ or Ca2+. Some capacity to bind guanine nucleotide is also retained by p21 treated with 7 M urea, as evidenced by increased resistance to proteolytic degradation, but the ability to bind divalent cations is irreversibly lost following denaturation. Protein prepared under denaturing conditions from a eukaryotic source, however, never regains the resistance to proteolysis shown by the bacterial p21 indicating irreversible changes in secondary and tertiary structure produced under these conditions.     

Action of extracellular divalent cations on native alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors

The effects of divalent cations on Ca2+-impermeable containing (GluR2 subunit) MPA receptors of hippocampal pyramidal neurones isolated from rat brain was studied using patch-clamping. Ca2+, Mg2+, Mn2+, Co2+, Ni2+ and Zn2+ inhibited currents induced by kainate and glutamate. Inhibition was fast, reversible and voltage independent. The rank order of activities was Ni2+ > Zn2+ > Co2+ > Ca2+ > Mn2+ > Mg2+. Cyclothiazide (0.1 mm) significantly reduced inhibition by divalent cations and 6, 7 dinitroquinoxaline-2.3-dione (DNQX). However, high concentrations of Ni2+ and DNQX inhibited AMPA receptors even in the presence of cyclothiazide. The inhibitory effect of divalent cations as well as DNQX was counteracted by an increase in agonist concentration. In the presence of divalent cations the EC50 values of kainate and glutamate were increased, but the maximal response was not changed. An increase in agonist concentration induced a parallel shift in the concentration-inhibition curve for a divalent cation. These data suggest a competitive-like type of inhibition. However, an increase in agonist concentration reduced the inhibitory action of Ni2+ less than that of DNQX. This gave evidence against direct competition between divalent cations and AMPA receptor agonists. A 'complex-competition' hypothesis was proposed to explain the inhibitory action of divalent cations; it is suggested that divalent cations form ion-agonist complexes, which compete with free agonist for agonist-binding sites on AMPA receptors.     

A calcium-binding, asparagine-linked oligosaccharide is involved in skeleton formation in the sea urchin embryo

We have previously identified a 130-kD cell surface protein that is involved in calcium uptake and skeleton formation by gastrula stage embryos of the sea urchin Strongylocentrotus purpuratus (Carson et al., 1985. Cell. 41:639-648). A monoclonal antibody designated mAb 1223 specifically recognizes the 130-kD protein and inhibits Ca+2 uptake and growth of the CaCO3 spicules produced by embryonic primary mesenchyme cells cultured in vitro. In this report, we demonstrate that the epitope recognized by mAb 1223 is located on an anionic, asparagine-linked oligosaccharide chain on the 130-kD protein. Combined enzymatic and chemical treatments indicate that the 1223 oligosaccharide contains fucose and sialic acid that is likely to be O-acetylated. Moreover, we show that the oligosaccharide chain containing the 1223 epitope specifically binds divalent cations, including Ca+2. We propose that one function of this negatively charged oligosaccharide moiety on the surfaces of primary mesenchyme cells is to facilitate binding and sequestration of Ca+2 ions from the blastocoelic fluid before internalization and subsequent deposition into the growing CaCO3 skeleton.     

Regulation by divalent cations of 3H-baclofen binding to GABAB sites in rat cerebellar membranes

When investigating the effects of divalent cations (Mg2+, Ca2+, Sr2+, Ba2+, Mn2+ and Ni2+) on 3H-baclofen binding to rat cerebellar synaptic membranes, we found that the specific binding of 3H-baclofen was not only dependent on divalent cations, but was increased dose-dependently in the presence of these cations. The effects were in the following order of potency: Mn2+ congruent to Ni2+ greater than Mg2+ greater than Ca2+ greater than Sr2+ greater than Ba2+. Scatchard analysis of the binding data revealed a single component of the binding sites in the presence of 2.5 mM MgCl2, 2.5 mM CaCl2 or 0.3 mM MnCl2 whereas two components appeared in the presence of 2.5 mM MnCl2 or 1 mM NiCl2. In the former, divalent cations altered the apparent affinity (Kd) without affecting density of the binding sites (Bmax). In the latter, the high-affinity sites showed a higher affinity and lower density of the binding sites than did the single component of the former. As the maximal effects of four cations (Mg2+, Ca2+, Mn2+ and Ni2+) were not additive, there are probably common sites of action of these divalent cations. Among the ligands for GABAB sites, the affinity for (-), (+) and (+/-) baclofen, GABA and beta-phenyl GABA increased 2-6 fold in the presence of 2.5 mM MnCl2, in comparison with that in HEPES-buffered Krebs solution (containing 2.5 mM CaCl2 and 1.2 mM MgSO4), whereas that for muscimol was decreased to one-fifth. Thus, the affinity of GABAB sites for its ligands is probably regulated by divalent cations, through common sites of action.     

Activation by divalent cations of a Ca2+-activated K+ channel from skeletal muscle membrane

Several divalent cations were studied as agonists of a Ca2+-activated K+ channel obtained from rat muscle membranes and incorporated into planar lipid bilayers. The effect of these agonists on single-channel currents was tested in the absence and in the presence of Ca2+. Among the divalent cations that activate the channel, Ca2+ is the most effective, followed by Cd2+, Sr2+, Mn2+, Fe2+, and Co2+. Mg2+, Ni2+, Ba2+, Cu2+, Zn2+, Hg2+, and Sn2+ are ineffective. The voltage dependence of channel activation is the same for all the divalent cations. The time-averaged probability of the open state is a sigmoidal function of the divalent cation concentration. The sigmoidal curves are described by a dissociation constant K and a Hill coefficient N. The values of these parameters, measured at 80 mV are: N = 2.1, K = 4 X 10(-7) mMN for Ca2+; N = 3.0, K = 0.02 mMN for Cd2+; N = 1.45, K = 0.63 mMN for Sr2+; N = 1.7, K = 0.94 mMN for Mn2+; N = 1.1, K = 3.0 mMN for Fe2+; and N = 1.1 K = 4.35 mMN for Co2+. In the presence of Ca2+, the divalent cations Cd2+, Co2+, Mn2+, Ni2+, and Mg2+ are able to increase the apparent affinity of the channel for Ca2+ and they increase the Hill coefficient in a concentration-dependent fashion. These divalent cations are only effective when added to the cytoplasmic side of the channel. We suggest that these divalent cations can bind to the channel, unmasking new Ca2+ sites.     

Hyaluronidase isoforms from developing embryos of the camel tick Hyalomma dromedarii

Changes in hyaluronidase activity in the camel tick Hyalomma dromedarii were followed throughout embryogenesis. Peak activity of the enzyme on days 21 and 24 during development was accompanied with a complete organization of larvae before hatching on day 27. During purification of hyaluronidase to homogeneity, ion exchange chromatography lead to four forms (HAase1, 2, 3 and 4). HAase2 and HAase4 with highest purity and specific activities after chromatography on Sephacryl S-200. The apparent molecular masses of HAase2 and HAase4 were 25 and 40 kDa, respectively. HAase2 and HAase4 had the same pH optimum of 3.6 and Km values of 0.3 and 0.34 mg/mL hyaluronic acid, respectively. Cleaving activities of HAase2 and HAase4 were demonstrated in the order: hyaluronic acid>chondroitin sulphate A>chondroitin sulphate C>chondroitin sulphate mixed>chondroitin sulphate B>heparin, low M.Wt>heparin. HAase2 and HAase4 had the same temperature optimum (40 degrees C) with heat stability up to 40 degrees C. H. dromedarii HAase2 and HAase4 had broad plateau of NaCl requirement with optimum activity recorded at 0.15 and 0.3 M NaCl, respectively. HAase2 and HAase4 were inhibited by Ca2+, Fe3+, Co2+ and Hg2+ and enhanced by Mg2+ and Mn2+.     

The effect of divalent cations on bovine spermatozoal adenylate cyclase activity.

The effect of divalent cations on bovine sperm adenylate cyclase activity was studied. Mn2+, Co2+, Cd2+, Zn2+, Mg2+ and Ca2+ were found to satisfy the divalent cation requirement for catalysis of the bovine sperm adenylate cyclase. These divalent cations in excess of the amount necessary for the formation of the metal-ATP substrate complex were found to stimulate the enzyme activity to various degrees. The magnitude of stimulation at saturating concentrations of the divalent cations was strikingly greater with M2+ than with either Ca2+, Mg2+, Zn2+, Cd2+ or Co2+. The apparent Km was lowest for Zm2+ (0.1 - 0.2 mM) than for any of the other divalent cations tested (1.2 - 2.3 mM). The enzyme stimulation by Mn2+ was decreased by the simultaneous addition of Co2+, Cd2+, Ni2+ and particularly Zn2+ and Cu2+. The antagonism between Mn2+ and Cu2+ or Zn2+ appeared to have both competitive and non-competitive features. The inhibitory effect of Cu2+ on Mn2+-stimulated adenylate cyclase activity was prevented by 2,3-dimercaptopropanol, but not by dithiothreitol, L-ergothioneine, EDTA, EGTA or D-penicillamine. Ca2+ at concentrations of 1-5 mM was found to act synergistically with Mg2+, Zn2+, Co2+ and Mn2+ in stimulating sperm adenylate cyclase activity. The Ca2+ augmentation of the stimulatory effect of Zn2+, Co2+, Mg2+ and Mn2+ appeared to be specific.     

Effect of divalent cations on the limited proteolysis of prothrombin by thrombin

The inhibitory influence of divalent cations on the ability of bovine alpha-thrombin to hydrolyze prothrombin showed the trend Mn2+ much greater than Ca2+ greater than or equal to Mg2+ greater than Sr2+ much greater than Ba2+. This effect was not due to an inhibition of thrombin's catalytic activity as measured by hydrolysis of a specific synthetic substrate, H-D-Phe-pipecolyl-Arg-p-nitroanilide (D-PhePipArgNA). The presence of divalent cations did not inhibit thrombic proteolysis of gamma-carboxyglutamic acid (Gla)-domainless prothrombin. Prothrombin and Gla-domainless prothrombin were used as competitive inhibitors in the thrombic hydrolysis of D-PhePipArgNA. The apparent Ki value calculated for prothrombin was 18 microM. When either Ca2+ or Mn2+ were present, there was no inhibition. The apparent Ki value determined for Gla-domainless prothrombin was 28 microM in either the absence or presence of Ca2+. Addition of divalent cations to prothrombin, but not to Gla-domainless prothrombin, resulted in an altered protein conformation as measured by high-performance size-exclusion chromatography and ultraviolet difference spectroscopy. These results suggest that a conformational change secondary to the interaction of divalent cations with the Gla-containing domain of prothrombin is required for cation-dependent inhibition of thrombin hydrolysis.     

The effect of divalent metal cations on the inhibition of human coagulation factor Xa by plasma proteinase inhibitors

The inactivation of human coagulation factor Xa by the plasma proteinase inhibitors alpha 1-antitrypsin, antithrombin III and alpha 2-macroglobulin in purified systems was found to be accelerated by the divalent cations Ca2+, Mn2+ and Mg2+. The rate constant for the inhibition of factor Xa by antithrombin III rose from 2.62 X 10(4) M-1 X min-1 in the absence of divalent cations to a maximum of 6.40 X 10(4) M-1 X min-1 at 5 mM Ca2+, 8.10 X 10(4) M-1 X min-1 at 5 mM Mn2+, with a slight decrease in rate at higher cation concentrations. Mg2+ caused a gradual rise in rate constant to 5.65 X 10(4) M-1 X min-1 at 20 mM. The rate constant for the inhibition of factor Xa by alpha 1-antitrypsin in the absence of divalent cations was 5.80 X 10(3) M-1 X min-1. Ca2+ increased the rate to 1.50 X 10(4) M-1 X min-1 at 5 mM and Mn2+ to 2.40 X 10(4) M-1 X min-1 at 6 mM. The rate constant for these cations again decreased at higher concentrations. Mg2+ caused a gradual rise in rate constant to 1.08 X 10(4) M-1 X min-1 at 10 mM. The rate constant for the factor Xa-alpha 2-macroglobulin reaction was raised from 6.70 X 10(3) M-1 X min-1 in the absence of divalent cations to a maximum of 4.15 X 10(4) M-1 X min-1 at 4 mM Ca2+, with a decrease to 3.05 X 10(4) M-1 at 10 mM. These increases in reaction rate were correlated to the binding of divalent cations to factor Xa by studying changes in the intrinsic fluorescence and dimerization of factor Xa. The changes in fluorescence suggested a conformational change in factor Xa which may be responsible for the increased rate of reaction, whilst the decrease in rate constant at higher concentrations of Ca2+ and Mn2+ may be due to factor Xa dimerization.     

The effect of monovalent and divalent cations on the activity of Streptococcus lactis C10 pyruvate kinase.

The pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from Streptococcus lactis C10 had an obligatory requirement for both a monovalent cation and divalent cation. NH+4 and K+ activated the enzyme in a sigmoidal manner (nH =1.55) at similar concentrations, whereas Na+ and Li+ could only weakly activate the enzyme. Of eight divalent cations studied, only three (Co2+, Mg2+ and Mn2+) activated the enzyme. The remaining five divalent cations (Cu2+, Zn2+, Ca2+, Ni2+ and Ba2+) inhibited the Mg2+ activated enzyme to varying degrees. (Cu2+ completely inhibited activity at 0.1 mM while Ba2+, the least potent inhibitor, caused 50% inhibition at 3.2 mM). In the presence of 1 mM fructose 1,6-diphosphate (Fru-1,6-P2) the enzyme showed a different kinetic response to each of the three activating divalent cations. For Co2+, Mn2+ and Mg2+ the Hill interaction coefficients (nH) were 1.6, 1.7 and 2.3 respectively and the respective divalent cation concentrations required for 50% maximum activity were 0.9, 0.46 and 0.9 mM. Only with Mn2+ as the divalent cation was there significatn activity in the absence of Fru-1,6-P2. When Mn2+ replaced Mg2+, the Fru-1,6-P2 activation changed from sigmoidal (nH = 2.0) to hyperbolic (nH = 1.0) kinetics and the Fru-1,6-P2 concentration required for 50% maximum activity decreased from 0.35 to 0.015 mM. The cooperativity of phosphoenolpyruvate binding increased (nH 1.2 to 1.8) and the value of the phosphoenolpyruvate concentration giving half maximal velocity decreased (0.18 to 0.015 mM phosphoenolyruvate) when Mg2+ was replaced by Mn2+ in the presence of 1 mM Fru-1,6-P2. The kinetic response to ADP was not altered significantly when Mn2+ was substituted for Mg2+. The effects of pH on the binding of phosphoenolpyruvate and Fru-1,6-P2 were different depending on whether Mg2+ or Mn2+ was the divalent cation.     

Cyclic nucleotide-gated channels of rat olfactory receptor cells: divalent cations control the sensitivity to cAMP

cAMP-gated channels were studied in inside-out membrane patches excised from the apical cellular pole of isolated olfactory receptor cells of the rat. In the absence of divalent cations the dose-response curve of activation of patch current by cAMP had a KM of 4.0 microM at -50 mV and of 2.5 microM at +50 mV. However, addition of 0.2 or 0.5 mM Ca2+ shifted the KM of cAMP reversibly to the higher cAMP concentrations of 33 or 90 microM, respectively, at -50 mV. Among divalent cations, the relative potency for inducing cAMP affinity shifts was: Ca2+ > Sr2+ > Mn2+ > Ba2+ > Mg2+, of which Mg2+ (up to 3 mM) did not shift the KM at all. This potency sequence corresponds closely to that required for the activation of calmodulin. However, the Ca(2+)-sensitivity is lower than expected for a calmodulin-mediated action. Brief (60 s) transient exposure to 3 mM Mg2+, in the absence of other divalent cations, had a protective effect in that following washout of Mg2+, subsequent exposure to 0.2 mM Ca2+ no longer caused affinity shifts. This protection effect did not occur in intact cells and was probably a consequence of patch excision, possibly representing ablation of a regulatory protein from the channel cyclic nucleotide binding site. Thus, the binding of divalent cations, probably via a regulatory protein, controls the sensitivity of the cAMP-gated channels to cAMP. The influx of Ca2+ through these channels during the odorant response may rise to a sufficiently high concentration at the intracellular membrane surface to contribute to the desensitization of the odorant- induced response. The results also indicate that divalent cation effects on cyclic nucleotide-gated channels may depend on the sequence of pre-exposure to other divalent cations.     

Divalent cation conduction in the ryanodine receptor channel of sheep cardiac muscle sarcoplasmic reticulum

The conduction properties of the alkaline earth divalent cations were determined in the purified sheep cardiac sarcoplasmic reticulum ryanodine receptor channel after reconstitution into planar phospholipid bilayers. Under bi-ionic conditions there was little difference in permeability among Ba2+, Ca2+, Sr2+, and Mg2+. However, there was a significant difference between the divalent cations and K+, with the divalent cations between 5.8- and 6.7-fold more permeant. Single-channel conductances were determined under symmetrical ionic conditions with 210 mM Ba2+ and Sr2+ and from the single-channel current-voltage relationship under bi-ionic conditions with 210 mM divalent cations and 210 mM K+. Single-channel conductance ranged from 202 pS for Ba2+ to 89 pS for Mg2+ and fell in the sequence Ba2+ greater than Sr2+ greater than Ca2+ greater than Mg2+. Near-maximal single-channel conductance is observed at concentrations as low as 2 mM Ba2+. Single-channel conductance and current measurements in mixtures of Ba(2+)-Mg2+ and Ba(2+)-Ca2+ reveal no anomalous behavior as the mole fraction of the ions is varied. The Ca(2+)-K+ reversal potential determined under bi-ionic conditions was independent of the absolute value of the ion concentrations. The data are compatible with the ryanodine receptor channel acting as a high conductance channel displaying moderate discrimination between divalent and monovalent cations. The channel behaves as though ion translocation occurs in single file with at most one ion able to occupy the conduction pathway at a time.     

Proton transport is necessary for divalent metal cations release from deenergized mitochondria

The release of divalent cations (Ca2+ and Sr2+) from rat liver mitochondria after membrane depolarization with protonophore (carbonyl cyanide m-chlorophenyl hydrazone, CCCP), sodium azide and K(+)-ionophore (valinomycin) was studied. It is stated that membrane depolarization itself is not sufficient for cations release from mitochondrial matrix (provided that mitochondrial permeability transition pore is blocked by cyclosporin A). Complete delivering of divalent cations is observed only after protonophore (CCCP) addition to suspension of deenergized mitochondria. The data show that membrane permeabilisation to hydrogen ions (H+) is necessary for complete cation release from the mitochondrial matrix. The enhancement in K(+)-conductivity of mitochondrial membrane (by valinomycin), on the contrary, is not able to provide complete delivering of cations from mitochondria. It is shown that quantity of divalent metal cation released from mitochondria (depolarized and permeabilized for K+ as well) is proportional to the concentration of protonophore (but not K(+)-ionophore) introduced in the incubation medium. The data obtained lead to the conclusion that H(+)-permeabilization of the mitochondrial membrane is necessary for the complete release of Ca2+ and Sr2+ from mitochondria after membrane depolarization. The possible mechanism of divalent metal cations release from deenergized mitochondria is discussed.     

The role of specific cations in regulation of cyanobacterial glutamine synthetase

Purified glutamine synthetase from the cyanobacterium Anabaena cylindrica required a divalent cation for activity. Maximum biosynthetic activity required Mg2+ (25 mM when supplied alone). Co2+ and Mn2+ each supported up to 20% of this activity; 12 other cations tested were ineffective. At 2.5 - 10 mM Mg2+, 0.1 mM Co2+ or ethylene glycol-bis-(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA) stimulated GS activity to maximum rates; other divalent cations (particularly Mn2+) inhibited Mg2+-dependent activity. At 5 mM Mg2+ the Kappm for NH+4 (0.05 mM) was 20-fold lower than at 25 mM Mg2+; added Co2+ did not markedly alter this low Km for NH+4; this could be physiologically important.     

Self-association of a high molecular weight subfragment-2 of myosin induced by divalent metal ions

The effect of divalent cations on the self-association of high molecular weight subfragment-2 (long S-2) and low molecular weight subfragment-2 (short S-2) of rabbit skeletal muscle myosin has been investigated. In the presence of millimolar concentrations of Ca2+ or Mg2+ long S-2 associates at neutral pH to form ordered, high molecular weight aggregates whereas short S-2 does not associate. The association process is co-operative and results from binding two to four divalent cations within the light meromyosin-heavy meromyosin (LMM-HMM) hinge region of long S-2. Optical diffraction of electron micrographs of the long S-2 aggregates revealed several periodicities including reflections near 143 A. High molecular weight HMM showed a similar divalent metal induced self-association. Chymotryptic digestion studies of rod filaments reveal that cleavage within the LMM-HMM hinge is also strongly dependent on the presence of divalent cations. At pH 8, in the absence of divalent cations, the S-2 region appears to be displaced away from the filament backbone resulting in rapid proteolysis in the hinge domain. At high cation concentrations (greater than 10 mM) proteolytic cleavage is suppressed. A similar depression of the (substantially lower) hinge cleavage rate was also observed at neutral pH following addition of these divalent metal ions. Results suggest that binding of Mg2+ within the hinge domain under physiological conditions may act to lock the cross-bridge onto the thick filament surface in its resting-state orientation.     

Activation of three types of membrane currents by various divalent cations in identified molluscan pacemaker neurons

We investigated membrane currents activated by intracellular divalent cations in two types of molluscan pacemaker neurons. A fast and quantitative pressure injection technique was used to apply Ca2+ and other divalent cations. Ca2+ was most effective in activating a nonspecific cation current and two types of K+ currents found in these cells. One type of outward current was quickly activated following injections with increasing effectiveness for divalent cations of ionic radii that were closer to the radius of Ca2+ (Ca2+ greater than Cd2+ greater than Hg2+ greater than Mn2+ greater than Zn2+ greater than Co2+ greater than Ni2+ greater than Pb2+ greater than Sr2+ greater than Mg2+ greater than Ba2+). The other type of outward current was activated with a delay by Ca2+ greater than Sr2+ greater than Hg2+ greater than Pb2+. Mg2+, Ba2+, Zn2+, Cd2+, Mn2+, Co2+, and Ni2+ were ineffective in concentrations up to 5 mM. Comparison with properties of Ca2(+)-sensitive proteins related to the binding of divalent cations suggests that a Ca2(+)-binding protein of the calmodulin/troponin C type is involved in Ca2(+)-dependent activation of the fast-activated type of K+ current. Th sequence obtained for the slowly activated type is compatible with the effectiveness of different divalent cations in activating protein kinase C. The nonspecific cation current was activated by Ca2+ greater than Hg2+ greater than Ba2+ greater than Pb2+ greater than Sr2+, a sequence unlike sequences for known Ca2(+)-binding proteins.     

Thermodynamic parameters for the binding of divalent cations to gramicidin A incorporated into a lipid environment by Tl-205 nuclear magnetic resonance.

Thermodynamic parameters, enthalpy and entropy, for the binding of the divalent cations, Mg+2, Ca+2, Sr+2, Ba+2, and Cd+2, to gramicidin A, incorporated into lysophosphatidylcholine, have been determined using a combination of Tl-205 nuclear magnetic resonance spectroscopy and competition binding. The binding process is thermodynamically driven by the enthalpy and not the entropy. The enthalpy values are related to the process involving the transfer of cations from an aqueous environment to an amide environment. A comparison is made between the thermodynamic parameters for the binding of monovalent and divalent cations to gramicidin A to illustrate the channel blocking ability of the divalent cations with respect to monovalent cation transport.     

Effects of monovalent and divalent cations and of guanine nucleotides on binding of vasopressin to the rat mesenteric vasculature

The rat mesenteric vasculature contains high affinity binding sites specific for [3H]Arg8-vasopressin which mediate its vasoconstrictor action. We have investigated the in vitro effect of monovalent and divalent cations and guanine nucleotides on the interactions between [3H]Arg8-vasopressin and its receptor in this preparation. Binding was increased by divalent cations from fourfold in the presence of Mg2+ at 5 mM to ninefold in the presence of Mn2+ at 5 mM. The potency order of divalent cations to increase binding was Mn2+ greater than Co2+ greater than Ni2+ greater than Mg2+ greater than Ca2+ approximately equal to control without cations. Addition of Na2+ or other monovalent cations (K+, Li+, and NH4+) in the presence or absence of divalent cations reduced binding significantly. Analysis of saturation binding curves showed a single high affinity site. In the presence of 5 mM Mn2+, binding capacity (Bmax) increased to 139 +/- 23 fmol/mg protein. Receptor affinity was enhanced (KD decreased to 0.33 +/- 0.07 nM). In presence of 5 mM Mg2+ or 150 mM Na+, Bmax and affinity were reduced. The addition of 100 microM GTP or its nonhydrolyzable analogue, Gpp(NH)p, reduced receptor affinity in the presence of Mn2+ + Na+, Mg2+, and Mg2+ + Na+, but not in the presence of Mn2+ alone. Computer modeling of competition binding curves demonstrated that in contrast with saturation studies, the data were best explained by a two-site model with high affinity, low capacity sites and low affinity, high capacity sites. Mn2+ or Mn2+ + Na+ with or without guanine nucleotides resulted in a predominance of high affinity sites. GTP or Gpp(NH)p in the presence of Mg2+ or Mg2+ + Na+ induced a reduction of affinity of the high affinity binding sites and the number of these sites. In the presence of Mg2+ + Na+ and guanine nucleotides, high affinity sites were maximally decreased. An association kinetic study indicated that the association rate constant (K+1) was increased by divalent cations and reduced by guanine nucleotides, without change in the dissociation rate constant (K-1). The equilibrium dissociation constant (KD) calculated with these rate constants (K-1/K+1) was similar to that obtained in saturation experiments at steady state. Dissociation kinetics were biphasic, indicating the presence of two receptor states, one of high and one of low affinity, associated with a slow and a rapid dissociation rate. Cations and guanine nucleotides interact with one or more sites closely associated with vasopressin receptors, including possibly with a GTP-sensitive regulatory protein, to modulate receptor affinity for vasopressin.     

Single-channel, macroscopic, and gating currents from sodium channels in the squid giant axon.

Single-channel, macroscopic ionic, and macroscopic gating currents were recorded from the voltage-dependent sodium channel using patch-clamp techniques on the cut-open squid giant axon. To obtain a complete set of physiological measurements of sodium channel gating under identical conditions, and to facilitate comparison with previous work, comparison was made between currents recorded in the absence of extracellular divalent cations and in the presence of physiological concentrations of extracellular Ca2+ (10 mM) and Mg2+ (50 mM). The single-channel currents were well resolved when divalent cations were not included in the extracellular solution, but were decreased in amplitude in the presence of Ca2+ and Mg2+ ions. The instantaneous current-voltage relationship obtained from macroscopic tail current measurements similarly was depressed by divalents, and showed a negative slope-conductance region for inward current at negative potentials. Voltage dependent parameters of channel gating were shifted 9-13 mV towards depolarized potentials by external divalent cations, including the peak fraction of channels open versus voltage, the time constant of tail current decline, the prepulse inactivation versus voltage relationship, and the charge-voltage relationship for gating currents. The effects of divalent cations are consistent with open channel block by Ca2+ and Mg2+ together with divalent screening of membrane charges.     

Selection and characterization of a 7‐mer peptide binding to divalent cations

A 7‐mer peptide (S‐T‐L‐P‐L‐P‐P) that bound to various divalent cations was selected from a phage display peptide library. Isothermal calorimetric analysis revealed that the peptide bound to Pb²⁺, Cd²⁺, Hg²⁺, and Cu²⁺. Through the use of CD studies, no secondary structural changes were observed for the peptide upon binding to divalent cations. Ala scanning mutant peptides bound to Hg²⁺ with a reduced affinity. However, no single substitution was shown to affect the overall affinity. We suggest that Pro residues chelate divalent cations, while the structure formed by the peptide is also important for the binding process. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.