Inactivation of calcium channels in vascular smooth muscle myocytes

Many of the structural domains involved in Ca²⁺ channel (CACN) inactivation are also involved in determining their sensitivity to antagonist inhibition. We hypothesize that differences in inactivation properties and their structural determinants may suggest candidate domains as targets for the development of novel, selective antagonists. The characteristics of Ca²⁺ current (I_Ca) inactivation, steady-state inactivation (SSIN), and recovery from inactivation were studied in freshly dispersed smooth muscle cells from rabbit portal vein (RPV) using whole-cell, voltage-clamp methods. The time course of inactivation could be represented by two time constants. Increasing I_Ca by increasing [Ca²⁺]_o or with more negative holding potentials decreased both time constants. With Sr²⁺, Ba²⁺, or Na⁺ as the charge carrier, I_Ca inactivation was also represented by two time constants, both of which were larger than those found with Ca²⁺. With Ca²⁺, Sr²⁺, or Ba²⁺ as the charge carrier, both time constants had minimum values near the voltage associated with maximum current. When Na⁺ (140 mM) was the charge carrier, voltages for I_max (−20 mV) or τ_min (o mV) did not correspond. SSIN of I_Ca had a half-maximum voltage of −32±4 mV for Ca²⁺, −43 mV±5 mV for Sr²⁺, −41±5 mV for Ba²⁺, and −68±6 mV for Na⁺. The slope factor for SSIN per e-fold voltage change was 6.5±0.2 mV for Ca²⁺, 6.8±0.3 for Sr²⁺, and 6.6±0.2 for Ba²⁺, representing four equivalent charges. When Na⁺ or Li⁺ was the charge carrier, the slope factor was 13.5±0.7 mV, representing two equivalent charges. For I_Ca in rat left ventricular (rLV) myocytes, there was no difference in the slope factor of SSIN for Ca²⁺ and Na⁺. The rate of recovery of I_Ca from inactivation varied inversely with recovery voltage and was independent of the charge carrier. These results suggest that inactivation of I_Ca in PV myocytes possess an intrinsic voltage dependence that is modified by Ca²⁺. For RPV but not rLV I_Ca, the charge of the permeating ion confers the voltage-dependency of SSIN.     

The interaction of proteins with hydroxyapatite. III. Mechanism

The mechanism of protein binding to and elution from hydroxyapatite (HA) has been established. Binding occurs both by nonspecific attraction between protein positive charges and HA and by specific complexing of protein carboxyls with calcium loci on the mineral. Elution can take place either as the result of the nonspecific ion screening of charges or by the specific displacement of protein groups from sites on the column with which they had complexed.     

Effect of concanavalin A on Ca2+ binding, uptake and the Ca2+ ATPase of lymphocyte plasma membranes

Lymphocyte plasma membranes bind ⁴⁵Ca²⁺ with three affinity sites: K_Al = 4.0 . 10⁶ M^?1, K_A2 = 8.5 . 10⁴ M^?1 and K_A3 = 4.2 . 10² M^?1, and Ca²⁺ binding capacities are 0.10, 1.2 and 85 nmoles Ca²⁺/mg protein. In the presence of 15 μg/ml ConA the Ca²⁺ binding constants were K_A1 = 4.6 . 10⁶ M^?1, K_A2 = 4.4 . 10⁴ M^?1 and K_A3 = 4.2 . 10² M^?1. The Ca²⁺ binding capacity was increased by ConA, to 0.13, 2.4 and 91 nmoles/mg protein. The Ca²⁺ ATPase activity of lymphocyte membranes was increased by ConA from 1 to 2 μmol P/protein × h. The ⁴⁵Ca²⁺ uptake was stimulated by ConA and PHA to about 16 %.     

The effect of Pb2+ on the structure and hydroxyapatite binding properties of osteocalcin

Lead toxicity is a major environmental health problem in the United States. Bone is the major reservoir for body lead. Although lead has been shown to impair bone metabolism in animals and at the cellular level, the effect of Pb²⁺ at the molecular level is largely unknown. We have used circular dichroism (CD), and a hydroxyapatite binding assay to investigate the effect of Pb²⁺ on the structure and mineral binding properties of osteocalcin, a noncollagenous bone protein. The CD data indicate Pb²⁺ induces a similar structure in osteocalcin as Ca²⁺ but at 2 orders of magnitude lower concentration. These results were explained by the more than 4 orders of magnitude tighter binding of Pb²⁺ to osteocalcin (K_d=0.085 μM) than Ca²⁺ (K_d=1.25 mM). The hydroxyapatite binding assays show that Pb²⁺ causes an increased adsorption to hydroxyapatite, similar to Ca²⁺, but at 2–3 orders of magnitude lower concentration. Low Pb²⁺ levels (1 μM) in addition to physiological Ca²⁺ levels (1 mM) caused a significant (40%) increase in the amount of mineral bound osteocalcin as compared to 1 mM Ca²⁺ alone. These results suggest a molecular mechanism of Pb²⁺ toxicity where low Pb²⁺ levels can inappropriately perturb Ca²⁺ regulated processes. In-vivo, the increased mineral bound osteocalcin could play a role in the observed low bone formation rates and decreased bone density observed in Pb²⁺-intoxicated animals.     

Sorption of alkaline earth metal ions Ca2+ and Mg2+ on lyocell fibres

Ca²⁺ and Mg²⁺ content of cellulose fibres is of relevance for a wide range of applications e.g. textile processing, pulp/paper, food. Sorption of Ca²⁺ and Mg²⁺ ions were found on lyocell type regenerated cellulose fibres. Higher affinity was found for Ca²⁺ ions compared to Mg²⁺ ions. At pH 9, fibre saturation was observed at a calcium binding capacity of 18–20 mmol/kg. A carboxylic group content of 18 mmol COOH per kg fibre material was determined based on the Methylene Blue absorption. This indicates a 1:1 molar stoichiometry between the carboxylic groups present in the fibres and the bound Ca²⁺ ions. Thus it is proposed that the salt in fibre shows the general composition (Cell-O^? Ca²⁺ X^?), X^? being an anion bound in the salt to achieve charge neutrality.The sorption of Ca²⁺ also can be demonstrated by complex formation with 1,2-dihydroxy-9,10-anthraquinone (alizarin) which forms a red-violet Ca²⁺-complex. Colour fixation thus can be used as an indicator for the Ca²⁺-ions bound in the fibre.     

Structural Basis for Difference in Heat Capacity Increments for Ca Binding to Two α-Lactalbumins

Thermodynamic parameters for the unfolding of as well as for the binding of Ca²⁺ to goat α-lactalbumin (GLA) and bovine α-lactalbumin (BLA) are deduced from isothermal titration calorimetry in a buffer containing 10 mM Tris-HCl, pH 7.5 near 25°C. Among the different parameters available, the heat capacity increments (ΔC_p) offer the most direct information for the associated conformational changes of the protein variants. The ΔC_p values for the transition from the native to the molten globule state are rather similar for both proteins, indicating that the extent of the corresponding conformational change is nearly identical. However, the respective ΔC_p values for the binding of Ca²⁺ are clearly different. The data suggest that a distinct protein region is more sensitive to a Ca²⁺-dependent conformational change in BLA than is the case in GLA. By analysis of the tertiary structure we observed an extensive accumulation of negatively charged amino acids near the Ca²⁺-binding site of BLA. In GLA, the cluster of negative charges is reduced by the substitution of Glu-11 by Lys. The observed difference in ΔC_p values for the binding of Ca²⁺ is presumably in part related to this difference in charge distribution.     

Glutamate 90 at the Luminal Ion Gate of Sarcoplasmic Reticulum Ca2+-ATPase Is Critical for Ca2+ Binding on Both Sides of the Membrane

The roles of Ser⁷², Glu⁹⁰, and Lys²⁹⁷ at the luminal ends of transmembrane helices M1, M2, and M4 of sarcoplasmic reticulum Ca²⁺-ATPase were examined by transient and steady-state kinetic analysis of mutants. The dependence on the luminal Ca²⁺ concentration of phosphorylation by P_i (“Ca²⁺ gradient-dependent E2P formation”) showed a reduction of the apparent affinity for luminal Ca²⁺ in mutants with alanine or leucine replacement of Glu⁹⁰, whereas arginine replacement of Glu⁹⁰ or Ser⁷² allowed E2P formation from P_i even at luminal Ca²⁺ concentrations much too small to support phosphorylation in wild type. The latter mutants further displayed a blocked dephosphorylation of E2P and an increased rate of conversion of the ADP-sensitive E1P phosphoenzyme intermediate to ADP-insensitive E2P as well as insensitivity of the E2·BeF₃⁻ complex to luminal Ca²⁺. Altogether, these findings, supported by structural modeling, indicate that the E2P intermediate is stabilized in the mutants with arginine replacement of Glu⁹⁰ or Ser⁷², because the positive charge of the arginine side chain mimics Ca²⁺ occupying a luminally exposed low affinity Ca²⁺ site of E2P, thus identifying an essential locus (a “leaving site”) on the luminal Ca²⁺ exit pathway. Mutants with alanine or leucine replacement of Glu⁹⁰ further displayed a marked slowing of the Ca²⁺ binding transition as well as slowing of the dissociation of Ca²⁺ from Ca₂E1 back toward the cytoplasm, thus demonstrating that Glu⁹⁰ is also critical for the function of the cytoplasmically exposed Ca²⁺ sites on the opposite side of the membrane relative to where Glu⁹⁰ is located.     

Binding of L-[3H]Glutamate to Fresh or Frozen Synaptic Membrane and Postsynaptic Density Fractions Isolated from Cerebral Cortex and Cerebellum of Fresh or Frozen Canine Brain

Abstract: Synaptic membrane (SPM) and postsynaptic density (PSD) fractions isolated from cerebral cortex (CTX) and cerebellum (CL) of canine brain, either fresh or frozen and isolated from either fresh or frozen tissue, were found to contain L-[³H]glutamate binding sites. It was found that there was a concentration of L-glutamate binding sites in CTX-PSD and CL-PSD over the respective membrane fractions, and the B_max value of CL-PSD (92.0 pmol/mg protein) was about three times that of CTX-PSD (28.9 pmol/mg). The results, together with those of others, suggest that the thin CL-PSD are probably derived from the excitatory synapses in the molecular layer. The ion dependency of L-glutamate binding to canine CTX-SPM fraction was found to be similar to that reported for a rat brain SPM fraction: (a) Cl^? increased the number of L-glutamate binding sites and the effect was enhanced by Ca²⁺; Ca²⁺ alone had no significant effect; (b) the Cl^?/Ca²⁺ -sensitive binding sites were abolished by 2-amino-4-phosphonobutyrate (APB) or freezing and thawing: (c) the effect of Na⁺ ion was biphasic: low concentration of Na⁺ (< 5 mM) decreased Cl^?7Ca²⁺ -de-pendent L-glutamate binding sites, whereas at higher concentrations of Na⁺ the binding of glutamate was found to increase either in the presence or absence of Ca²⁺ and Cl^?. In addition, the K⁺ ion (50 mM) was found to decrease the Na⁺-independent and Cl^?/Ca^2--independent binding of L-glutamate to fresh CTX-SPM by 18%, but it decreased the Na^?-dependent and Cl^?/Ca²⁺-independent L-glutamate binding by 93%; in the presence of Cl, ^?/Ca²⁺, the K⁺ ion decreased the Na⁺-dependent binding by 78%. Freezing and thawing of CTX-SPM resulted in a 50% loss of the Na⁺-dependent L-glutamate binding sites assayed in the absence of Ca²⁺ and Cl^?. The CL-SPM fraction showed similar ion dependency of L-glutamate binding except for the absence of Na^?-dependent glutamate binding sites. The CTX-PSD fraction contained neither Na⁺-dependent nor APB (or Cl^?/Ca²⁺)-sensitive L-glutamate binding sites and its L-glutamate binding was unaffected by freezing and thawing, in agreement with the reported findings using rat brain PSD preparation. L-Glutamate binding to CTX-SPM or CTX-PSD fraction was not affected by pretreatment with 10 mM L-glutamate, nor by simultaneous incubations with calmodulin. Also, phosphorylation of CTX-SPM or CTX-PSD fraction, whether incubated simultaneously or after removal of the phosphorylating reagents, had no effect on binding of L-glutamate. Furthermore, binding of L-glutamate to CTX-SPM or CTX-PSD was found to have no significant effect on subsequent phosphorylation of the fractions. Treatment of the CTX-PSD fraction with 0.5% deoxycholate, 1.0% N-lauroyl sarcosinate, 4 M guanidine-HCl, pH 7.0, 0.5 M KCl, and 1.0 M KCl removed the L-glutamate receptors from the PSD by 25%, 44%, 40%, 8%, and 11%. respectively. The respective percentages of total protein solubilized by these reagents were similar, indicating no preferential dissociation of the receptors, and suggesting that the L-glutamate receptor is an intrinsic PSD component. The present findings, together with the earlier ones showing the presence of γ-aminobutyric acid and flunitrazepam binding sites, of the Ca²⁺-dependent K⁺ channel, and of the voltage-dependent Ca²⁺ channel proteins in the isolated PSD fraction, suggest that many, if not all, neurotransmitter receptor proteins and ion channel proteins are anchored in the PSD at the synapse, and thus the PSD may play an important role in neurotransmission at the postsynaptic site.     

Simultaneous Binding of Basic Peptides at Intracellular Sites on a Large Conductance Ca2+-activated K+ Channel : Equilibrium and Kinetic Basis of Negatively Coupled Ligand Interactions

The homologous Kunitz inhibitor proteins, bovine pancreatic trypsin inhibitor (BPTI) and dendrotoxin I (DTX-I), interact with large conductance Ca²⁺-activated K⁺ channels (maxi-K_Ca) by binding to an intracellular site outside of the pore to produce discrete substate events. In contrast, certain homologues of the Shaker ball peptide produce discrete blocking events by binding within the ion conduction pathway. In this study, we investigated ligand interactions of these positively charged peptide molecules by analysis of single maxi-K_Ca channels in planar bilayers recorded in the presence of DTX-I and BPTI, or DTX-I and a high-affinity homologue of ball peptide. Both DTX-I (K _d, 16.5 nM) and BPTI (K _d, 1,490 nM) exhibit one-site binding kinetics when studied alone; however, records in the presence of DTX-I plus BPTI demonstrate simultaneous binding of these two molecules. The affinity of BPTI (net charge, +6) decreases by 11.7-fold (K _d, 17,500 nM) when DTX-I (net charge, +10) is bound and, conversely, the affinity of DTX-I decreases by 10.8-fold (K _d, 178 nM) when BPTI is bound. The ball peptide homologue (BP; net charge, +6) exhibits high blocking affinity (K _d, 7.2 nM) at a single site when studied alone, but has 8.0-fold lower affinity (K _d, 57 nM) for blocking the DTX-occupied channel. The affinity of DTX-I likewise decreases by 8.4-fold (K _d, 139 nM) when BP is bound. These results identify two types of negatively coupled ligand–ligand interactions at distinct sites on the intracellular surface of maxi-K_Ca channels. Such antagonistic ligand interactions explain how the binding of BPTI or DTX-I to four potentially available sites on a tetrameric channel protein can exhibit apparent one-site kinetics. We hypothesize that negatively coupled binding equilibria and asymmetric changes in transition state energies for the interaction between DTX-I and BP originate from repulsive electrostatic interactions between positively charged peptide ligands on the channel surface. In contrast, there is no detectable binding interaction between DTX-I on the inside and tetraethylammonium or charybdotoxin on the outside of the maxi-K_Ca channel.     

Interfacial Kinetic and Binding Properties of Mammalian Group IVB Phospholipase A2 (cPLA2β) and Comparison with the Other cPLA2 Isoforms

The cytosolic (group IV) phospholipase A₂ (cPLA₂s) family contains six members. We have prepared recombinant proteins for human α, mouse β, human γ, human δ, human ϵ, and mouse ζ cPLA₂s and have studied their interfacial kinetic and binding properties in vitro. Mouse cPLA₂β action on phosphatidylcholine vesicles is activated by anionic phosphoinositides and cardiolipin but displays a requirement for Ca²⁺ only in the presence of cardiolipin. This activation pattern is explained by the effects of anionic phospholipids and Ca²⁺ on the interfacial binding of mouse cPLA₂β and its C2 domain to vesicles. Ca²⁺-dependent binding of mouse cPLA₂β to cardiolipin-containing vesicles requires a patch of basic residues near the Ca²⁺-binding surface loops of the C2 domain, but binding to phosphoinositide-containing vesicles does not depend on any specific cluster of basic residues. Human cPLA₂δ also displays Ca²⁺- and cardiolipin-enhanced interfacial binding and activity. The lysophospholipase, phospholipase A₁, and phospholipase A₂ activities of the full set of mammalian cPLA₂s were quantified. The relative level of these activities is very different among the isoforms, and human cPLA₂δ stands out as having relatively high phospholipase A₁ activity. We also tested the susceptibility of all cPLA₂ family members to a panel of previously reported inhibitors of human cPLA₂α and analogs of these compounds. This led to the discovery of a potent and selective inhibitor of mouse cPLA₂β. These in vitro studies help determine the regulation and function of the cPLA₂ family members.     

Resolution and characterization of two soluble calcium-dependent protein kinases from silver beet leaves

Two soluble Ca²⁺-dependent protein kinases (enzymes I and II) have been extensively purified from silver beet leaf tissue by means of a protocol involving batch-wise elution from DEAE-cellulose, Ca²⁺-dependent binding to phenyl-Sepharose, gradient elution from DEAE-Sephacel, gel filtration and binding to Cibacron F3GA-Sepharose CL-6B. Protein kinases I and II are resolved on gradient elution from DEAE-Sephacel and are further distinguished by their different K_m values for ATP and large differences in relative rates of phosphorylation of histone H1, casein and bovine serum albumin (the latter two proteins are relatively poor substrates for enzyme II but not enzyme I). Both enzymes have similar molecular weights as determined from gel filtration (56000 ± 2000 and 57000 ± 3000 for enzymes I and II, respectively). Both enzymes are absolutely dependent on free Ca²⁺ for activity with maximal histone H1 kinase activity being obtained at 0.5 μM free Ca²⁺. A millimolar concentration of Mg²⁺ is required in addition to a micromolar concentration Ca²⁺ for maximal activity. Both enzymes specifically phosphorylate serine residues of histone H1, are thiol activated and are inhibited by lanthanides and a range of calmodulin antagonists and inhibitors of protein kinase C.     

Inorganic speciation of organotin(IV) cations in natural waters with particular reference to seawater

Abstract

This paper examines the inorganic complexing capacity of seawater, where chloride and sulfate ions are present in high concentration, towards mono- di- and tri-organotin(IV) cations which show a different trend of acidity, depending on cation charges, and a corresponding tendency to hydrolysis. By considering hydrolytic species and chloride and sulphate complex formation, a basic inorganic speciation model of organotins in synthetic seawater (Na⁺, K⁺, Ca²⁺, Mg²⁺, Cl^?, SO₄^2?) has been built up. The model has been extended to also consider interactions of organotins with carbonate and fluoride ions, which are other important components of seawater. Because of the strength of hydrolysis processes, the main complexes formed are in general mixed hydroxo-species. No species are formed by organotin cations and/or their hydroxo-species with fluoride owing to their very low concentration in fluoride, in comparison to the other components of seawater. In order to simplify calculations and to establish a cumulative inorganic binding capacity for seawater, we applied a chemical complexation model, according to which the major inorganic components of seawater are considered as a single salt BA.     

Probing Calcium Ion-Induced Conformational Changes of Taiwan Cobra Phospholipase A2 by Trinitrophenylation of Lysine Residues

Phospholipase A₂ (PLA₂) from Naja naja atra (Taiwan cobra) snake venom was subjected to lysine modification with trinitrobenzene sulfonate (TNBS). Three major derivatives, TNP-1, TNP-2, and TNP-3, were separated by high-performance liquid chromatography (HPLC) from the reaction mixtures in the absence of Ca²⁺. However, only TNP-2 and TNP-3 were isolated when trinitrophenylated reaction was carried out in the presence of Ca²⁺. TNP-1 and TNP-2 contained only one TNP group, on Lys-65 and Lys-6, respectively; and both Lys-6 and Lys-65 were modified in TNP-3. The extent of modification on Lys-6 and Lys-65 was calculated from the peak areas of TNP proteins in the HPLC profile. It was found that the susceptibility of Lys-6 toward TNBS markedly increased by the addition of Ca²⁺ when Ca²⁺ concentration was higher than 5 mM. With regard to the involvement of Lys-6 in the binding of substrate, the increase in the reactivity of Lys-6 may arise from a conformational change around Lys-6 for binding with substrate in the presence of Ca²⁺. Alternatively, the nonessentiality of Lys-65 for PLA₂ activity was revealed by the finding that TNP-1 still retained 95% activity of native enzyme. Moreover, the reactivity of Lys-65 toward TNBS did not greatly change in either the absence or presence of Ca²⁺, suggesting that Ca²⁺ binding did not cause an appreciable change in the microenvironment around Lys-65. These results indicate that the differential reactivities of Lys-6 and Lys-65 toward TNBS as affected by the binding of Ca²⁺ are well consistent with their functional roles in the catalytic mechanism of PLA₂, and suggest that the occurrence of conformational changes with PLA₂ could be explored by chemical modification studies.     

The calcium requirement in GnRH-stimulated LH release is not mediated through a specific action on receptor binding

A radioligand receptor assay was used to determine the effect of Ca⁺² and other cations on the binding of D-Ser-(t-Bu)⁶-desGly¹⁰EA GnRH (Buserelin acetate, Hoechst) to the gonadotropin releasing hormone receptor in rat pituitary membrane. Specific binding was reduced in the presence of CaCl₂, BaCl₂, MnCl₂, Co(CH₃COO)₂, MgCl₂, NaCl, or LaCl₃, although these salts have markedly different effects on receptor mediated gonadotropin release from intact cells. Consistent with this finding, compounds which chelate cations increased specific binding of the ligand. The decreased binding is explained solely by an effect on receptor affinity, which was reduced from 4 × 10⁹ M^?1 in the presence of 10 mM CaCl₂. Total receptor number was unchanged. These data suggest that the requirement for Ca⁺² in GnRH-stimulated LH release is not mediated through a specific action of this ion at the level of receptor binding.     

Probing Calcium Ion-Induced Conformational Changes of Taiwan Cobra Phospholipase A2 by Trinitrophenylation of Lysine Residues

Phospholipase A₂ (PLA₂) from Naja naja atra (Taiwan cobra) snake venom was subjected to lysine modification with trinitrobenzene sulfonate (TNBS). Three major derivatives, TNP-1, TNP-2, and TNP-3, were separated by high-performance liquid chromatography (HPLC) from the reaction mixtures in the absence of Ca²⁺. However, only TNP-2 and TNP-3 were isolated when trinitrophenylated reaction was carried out in the presence of Ca²⁺. TNP-1 and TNP-2 contained only one TNP group, on Lys-65 and Lys-6, respectively; and both Lys-6 and Lys-65 were modified in TNP-3. The extent of modification on Lys-6 and Lys-65 was calculated from the peak areas of TNP proteins in the HPLC profile. It was found that the susceptibility of Lys-6 toward TNBS markedly increased by the addition of Ca²⁺ when Ca²⁺ concentration was higher than 5 mM. With regard to the involvement of Lys-6 in the binding of substrate, the increase in the reactivity of Lys-6 may arise from a conformational change around Lys-6 for binding with substrate in the presence of Ca²⁺. Alternatively, the nonessentiality of Lys-65 for PLA₂ activity was revealed by the finding that TNP-1 still retained 95% activity of native enzyme. Moreover, the reactivity of Lys-65 toward TNBS did not greatly change in either the absence or presence of Ca²⁺, suggesting that Ca²⁺ binding did not cause an appreciable change in the microenvironment around Lys-65. These results indicate that the differential reactivities of Lys-6 and Lys-65 toward TNBS as affected by the binding of Ca²⁺ are well consistent with their functional roles in the catalytic mechanism of PLA₂, and suggest that the occurrence of conformational changes with PLA₂ could be explored by chemical modification studies.     

The mobility of troponin C and troponin I in muscle

In vertebrate skeletal muscle, contraction is initiated by the elevation of the intracellular Ca²⁺ concentration. The binding of Ca²⁺ to TnC induces a series of conformational changes which ultimately release the inhibition of the actomyosin ATPase activity by Tnl. In this study we have characterized the dynamic behavior of TnC and Tnl in solution, as well as in reconstituted fibers, using EPR and ST-EPR spectroscopy. Cys98 of TnC and Cys133 of Tnl were specifically labeled with malemide spin label (MSL) and indane dione nitroxide spin label (InVSL). In solution, the labeled TnC and Tnl exhibited fast nanosecond motion. MSL-TnC is sensitive to cation binding to the high affinity sites (τ_r increases from 1.5 to 3.7 ns), InVSL-TnC s sensitive to the replacement of Mg²⁺ by Ca²⁺ at these sites (τ_r increase from 1.7 to 6 ns). Upon reconstitution into fibers, the nanosecond mobility is reduced by interactions with other proteins. TnC and Tnl both exhibited microsecond anisotropic motion in fibers similar to that of the actin monomers within the filament. The microsecond motion of TnC was found to be modulated by the binding of Ca²⁺ and by cross-bridge attachment, but this was not the case for the global mobility of Tnl. © 1997 John Wiley & Sons, Ltd.     

Theoretical description of the calcium channel in rat spinal ganglion neurons

The surface charge density (σ′₀) and the binding constant of Ca⁺⁺ with charged groups on the outer surface of the membrane (K_Ca) were calculated from experimentally determined values of the shift of the current-voltage characteristic curves of calcium currents in the membrane of rat spinal ganglion neurons: σ′₀ = 0.15 ± 0.05 e/nm² and K_Ca = 70 ± 10 liters/mole. Using a three-barrier model the energy profile of the calcium channel of the membrane of these neurons was calculated for Ca⁺⁺, Ba⁺⁺, Cd⁺⁺, Mn⁺⁺, Co⁺⁺, and verapamil. The calcium current was shown to be determined mainly by the depth of the potential hole corresponding to the outer binding site of the calcium channel. It is concluded from the results that the outer binding site of the calcium channel contains only one carboxyl group.     

Significance of Troponin Dynamics for Ca2+-mediated Regulation of Contraction and Inherited Cardiomyopathy

Ca²⁺ dissociation from troponin causes cessation of muscle contraction by incompletely understood structural mechanisms. To investigate this process, regulatory site Ca²⁺ binding in the NH₂-lobe of subunit troponin C (TnC) was abolished by mutagenesis, and effects on cardiac troponin dynamics were mapped by hydrogen-deuterium exchange (HDX)-MS. The findings demonstrate the interrelationships among troponin''s detailed dynamics, troponin''s regulatory actions, and the pathogenesis of cardiomyopathy linked to troponin mutations. Ca²⁺ slowed HDX up to 2 orders of magnitude within the NH₂-lobe and the NH₂-lobe-associated TnI switch helix, implying that Ca²⁺ greatly stabilizes this troponin regulatory region. HDX of the TnI COOH terminus indicated that its known role in regulation involves a partially folded rather than unfolded structure in the absence of Ca²⁺ and actin. Ca²⁺-triggered stabilization extended beyond the known direct regulatory regions: to the start of the nearby TnI helix 1 and to the COOH terminus of the TnT-TnI coiled-coil. Ca²⁺ destabilized rather than stabilized specific TnI segments within the coiled-coil and destabilized a region not previously implicated in Ca²⁺-mediated regulation: the coiled-coil''s NH₂-terminal base plus the preceding TnI loop with which the base interacts. Cardiomyopathy-linked mutations clustered almost entirely within influentially dynamic regions of troponin, and many sites were Ca²⁺-sensitive. Overall, the findings demonstrate highly selective effects of regulatory site Ca²⁺, including opposite changes in protein dynamics at opposite ends of the troponin core domain. Ca²⁺ release triggers an intramolecular switching mechanism that propagates extensively within the extended troponin structure, suggests specific movements of the TnI inhibitory regions, and prominently involves troponin''s dynamic features.     

Binding and Functional Folding (BFF): A Physiological Framework for Studying Biomolecular Interactions and Allostery

EF-hand Ca²⁺-binding proteins (CBPs), such as S100 proteins (S100s) and calmodulin (CaM), are signaling proteins that undergo conformational changes upon increasing intracellular Ca²⁺. Upon binding Ca²⁺, S100 proteins and CaM interact with protein targets and induce important biological responses. The Ca²⁺-binding affinity of CaM and most S100s in the absence of target is weak (^CaK_D > 1 μM). However, upon effector protein binding, the Ca²⁺ affinity of these proteins increases via heterotropic allostery (^CaK_D < 1 μM). Because of the high number and micromolar concentrations of EF-hand CBPs in a cell, at any given time, allostery is required physiologically, allowing for (i) proper Ca²⁺ homeostasis and (ii) strict maintenance of Ca²⁺-signaling within a narrow dynamic range of free Ca²⁺ ion concentrations, [Ca²⁺]^free. In this review, mechanisms of allostery are coalesced into an empirical “binding and functional folding (BFF)” physiological framework. At the molecular level, folding (F), binding and folding (BF), and BFF events include all atoms in the biomolecular complex under study. The BFF framework is introduced with two straightforward BFF types for proteins (type 1, concerted; type 2, stepwise) and considers how homologous and nonhomologous amino acid residues of CBPs and their effector protein(s) evolved to provide allosteric tightening of Ca²⁺ and simultaneously determine how specific and relatively promiscuous CBP-target complexes form as both are needed for proper cellular function.     

A Long-Term Blockade of L-Type Calcium Currents Upregulates the Number of Ca2+ Channels in Skeletal Muscle

The effects of a long-term blockade of L-type Ca²⁺ channels on membrane currents and on the number of dihydropyridine binding sites were investigated in skeletal muscle fibers. Ca²⁺ currents (I _Ca) and intramembrane charge movement were monitored using a voltage-clamp technique. The peak amplitude of I _Ca increased by more than 40% in fibers that were previously incubated for 24 hr in solutions containing the organic Ca²⁺ channel blocker nifedipine or in Ca²⁺-free conditions. A similar incubation period with Cd²⁺, an inorganic blocker, produced a moderate increase of 20% in peak I _Ca. The maximum mobilized charge (Q _max) increased by 50% in fibers preincubated in Ca²⁺-free solutions or in the presence of Cd²⁺. Microsomal preparations from frog skeletal muscle were isolated by differential centrifugation. Preincubation with Cd²⁺ prior to the isolation of the microsomal fraction doubled the number of ³H-PN200-110 binding sites and produced a similar increase in the values of the dissociation constant. The increase in the number of binding sites is consistent with the increase in the peak amplitude of I _Ca as well as with the increase in Q _max. Received: 31 August 1998/Revised: 7 December 1998