The Effects of Solvent Environment on the Optical Rotatory Dispersion Parameters of Polypeptides: II. Studies on Poly-l-Glutamic Acid

The Moffitt b₀ parameter of poly-L-glutamic acid in the presumed helical state varied with solvent composition, ranging in magnitude from less than 600° in aqueous solution to 800° in methanol. b₀ was also dependent on temperature throughout the excessable temperature range. The value in aqueous solution is at least 100° smaller than the values for a number of polypeptides in organic solvents, when compared at the same refractive index. Therefore the optical rotatory dispersion data do not provide evidence that the molecule is completely helical in aqueous solution. Since other types of evidence for helical content are not sufficient to establish that PLGA is a complete helix, the helical content of proteins and polypeptides determined by rotatory dispersion measurements should be regarded as uncertain by about 20 per cent.     

Individual Interactions of the b Subunits within the Stator of the Escherichia coli ATP Synthase

F_OF₁ ATP synthases are rotary nanomotors that couple proton translocation across biological membranes to the synthesis/hydrolysis of ATP. During catalysis, the peripheral stalk, composed of two b subunits and subunit δ in Escherichia coli, counteracts the torque generated by the rotation of the central stalk. Here we characterize individual interactions of the b subunits within the stator by use of monoclonal antibodies and nearest neighbor analyses via intersubunit disulfide bond formation. Antibody binding studies revealed that the C-terminal region of one of the two b subunits is principally involved in the binding of subunit δ, whereas the other one is accessible to antibody binding without impact on the function of F_OF₁. Individually substituted cysteine pairs suitable for disulfide cross-linking between the b subunits and the other stator subunits (b-α, b-β, b-δ, and b-a) were screened and combined with each other to discriminate between the two b subunits (i.e. b_I and b_II). The results show the b dimer to be located at a non-catalytic α/β cleft, with b_I close to subunit α, whereas b_II is proximal to subunit β. Furthermore, b_I can be linked to subunit δ as well as to subunit a. Among the subcomplexes formed were a-b_I-α, b_II-β, α-b_I-b_II-β, and a-b_I-δ. Taken together, the data obtained define the different positions of the two b subunits at a non-catalytic interface and imply that each b subunit has a different role in generating stability within the stator. We suggest that b_I is functionally related to the single b subunit present in mitochondrial ATP synthase.     

Solvent and solute effects on “inside-outside” conformations and rotation about the peptide bond in a miniature protein model

The physicochemical parameters affecting protein unfolding in relation to peptide bond rotations are briefly reviewed. As a suitable model for the study of solvent and solute effects on amide rotation and inside-outside conformations, the 2,2′-biphenyl analog of N-benzoyl-l-phenylalanine methyl ester (I) was synthesized and resolved enzymatically with α-chymotrypsin. The optically pure substance exists as conformer I_a (R,S configuration) with an axial methoxycarbonyl in the crystalline state. Rotation about the biphenyl axis leads to the equatorial conformer I_b (S,S configuration) in various solvents. In polar solvents, rotation about the amide is rate limiting. Accurate measurements of this rotation were accomplished by following the rate of change in the maximum amplitude of the biphenyl Cotton band at 256 nm. The high sensitivity of the method allowed rate and equilibrium measurements at 10^?3M in the absence of intermolecular association. Small differences of the order of 100 cal/mole in ΔG^≠ or ΔG_eq could thus be detected accurately. It was found that k_obs or k₁ (forward step) for equilibration was linearly related (correlation coefficient of 0.96 for k_obs) with E_T, the solvent polarity index on Reichardt and Dimroth's scale. Rotation was slowest in water and fastest in carbon tetrachloride, δΔG^≠, being 2.4 kcal/mole. Chaotropic anions, cations, and guanidinium chloride accelerated the rate in water. However, the inside-outside (axial-equatorial; I_aI_b) ratio at equilibrium did not correlate in any simple manner with the solvent E_T values. Rather, correlation within groups of solvents appeared to exist. It was suggested that solvent association with the amide differs quantitatively in the inside and outside conformations. The position of the equilibrium in water was affected by chaotropic ions but not by urea or quanidinium chloride. Some possible mechanisms are briefly outlined.     

Magnetic Resonance Water Proton Relaxation in Protein Solutions and Tissue: T1ρ Dispersion Characterization

Background

Image contrast in clinical MRI is often determined by differences in tissue water proton relaxation behavior. However, many aspects of water proton relaxation in complex biological media, such as protein solutions and tissue are not well understood, perhaps due to the limited empirical data.

Principal Findings

Water proton T₁, T₂, and T_1ρ of protein solutions and tissue were measured systematically under multiple conditions. Crosslinking or aggregation of protein decreased T₂ and T_1ρ, but did not change high-field T₁. T_1ρ dispersion profiles were similar for crosslinked protein solutions, myocardial tissue, and cartilage, and exhibited power law behavior with T_1ρ(0) values that closely approximated T₂. The T_1ρ dispersion of mobile protein solutions was flat above 5 kHz, but showed a steep curve below 5 kHz that was sensitive to changes in pH. The T_1ρ dispersion of crosslinked BSA and cartilage in DMSO solvent closely resembled that of water solvent above 5 kHz but showed decreased dispersion below 5 kHz.

Conclusions

Proton exchange is a minor pathway for tissue T₁ and T_1ρ relaxation above 5 kHz. Potential models for relaxation are discussed, however the same molecular mechanism appears to be responsible across 5 decades of frequencies from T_1ρ to T₁.     

Propagation Speed in Myelinated Nerve: II. Theoretical Dependence on External Na+ and on Temperature

The Hodgkin-Huxley (H.H.) equations modified by Dodge for Rana pipiens myelinated nerve have been solved to determine how well the theory predicts the effects of changes of temperature and [Na⁺]₀ on propagation. Conduction speed θ was found to have an approximately exponential dependence on temperature as was found experimentally, but the theoretical temperature coefficient (Q₁₀) was low; 1.5 compared with the experimental finding of 2.95. θ was found to be a linear function of log ([Na⁺]₀) in contrast to the experimental finding of a square root dependence on [Na⁺]₀. θ is 50% greater at one-fourth normal [Na⁺]₀ than the theory predicts. The difference between the theoretical θ([Na⁺]₀) and the experimental θ([Na⁺]₀) is probably due to an imprecisely known variation of parameters and not to a fundamental inadequacy of the theory.     

Quantitative models characterizing seed germination responses to abscisic Acid and osmoticum

Mathematical models were developed to characterize the physiological bases of the responses of tomato (Lycopersicon esculentum Mill. cv T5) seed germination to water potential (ψ) and abscisic acid (ABA). Using probit analysis, three parameters were derived that can describe the germination time courses of a seed population at different ψ or ABA levels. For the response of seed germination to reduced ψ, these parameters are the mean base water potential (¯ψ_b, MPa), the standard deviation of the base water potential among seeds in the population (σ_ψb, MPa), and the “hydrotime constant” (θ_H, MPa·h). For the response to ABA, they are the log of the mean base ABA concentration ([unk]ABA_b, m), the standard deviation of the base ABA concentration among seeds in the population (σ_ABAb, log[m]), and the “ABA-time constant” (θ_ABA, log[m]·h). The values of ¯ψ_b and [unk]ABA_b provide quantitative estimates of the mean sensitivity of germination rate to ψ or ABA, whereas σ^ψ_b and σ_ABAb account for the variation in sensitivity among seeds in the population. The time constants, θ_H and θ_ABA, indicate the extent to which germination rate will be affected by a given change in ψ or ABA. Using only these parameters, germination time courses can be predicted with reasonable accuracy at any medium ψ according to the equation probit(g) = [ψ - (θ_H/t_g) - ¯ψ_b]/σ_ψb, or at any ABA concentration according to the equation probit(g) = [log[ABA] - (θ_ABA/t_g) - log[[unk]ABA_b]]/σ_ABAb, where t_g is the time to radicle emergence of percentage g, and ABA is the ABA concentration (m) in the incubation solution. In the presence of both ABA and reduced ψ, the same parameters can be used to predict seed germination time courses based upon strictly additive effects of ψ and ABA in delaying the time of radicle emergence. Further analysis indicates that ABA and ψ can act both independently and interactively to influence physiological processes preparatory for radicle growth, such as the accumulation of osmotic solutes in the embryo. The models provide quantitative values for the sensitivity of germination to ABA or ψ, allow evaluation of independent and interactive effects of the two factors, and have implications for understanding how ABA and ψ may regulate growth and development.     

Purification of Extracellular Cholesterol Oxidase with High Activity in the Presence of Organic Solvents from Pseudomonas sp. Strain ST-200

Extracellular cholesterol oxidase of Pseudomonas sp. strain ST-200 was purified from the culture supernatant. This oxidase contained bound flavin and was categorized as a 3β-hydroxysteroid oxidase, converting 3β-hydroxyl groups to keto groups. The molecular mass was 60 kDa. The enzyme was stable at pH 4 to 11 and active at pH 5.0 to 8.5, showing optimal activity at pH 7 at 60°C. The Michaelis constant of the ST-200 cholesterol oxidase was lower than those of commercially available oxidases. The cholesterol oxidation rate was enhanced 3- to 3.5-fold in the presence of organic solvents, with log P_ow values (partition coefficients of the organic solvent between n-octanol and water), in the range of 2.1 to 4.2, compared with that in the absence of organic solvents.     

Solvent Effect on the Photolysis of Riboflavin

The kinetics of photolysis of riboflavin (RF) in water (pH 7.0) and in organic solvents (acetonitrile, methanol, ethanol, 1-propanol, 1-butanol, ethyl acetate) has been studied using a multicomponent spectrometric method for the assay of RF and its major photoproducts, formylmethylflavin and lumichrome. The apparent first-order rate constants (k_obs) for the reaction range from 3.19 (ethyl acetate) to 4.61 × 10⁻³ min⁻¹ (water). The values of k_obs have been found to be a linear function of solvent dielectric constant implying the participation of a dipolar intermediate along the reaction pathway. The degradation of this intermediate is promoted by the polarity of the medium. This indicates a greater stabilization of the excited-triplet states of RF with an increase in solvent polarity to facilitate its reduction. The rate constants for the reaction show a linear relation with the solvent acceptor number indicating the degree of solute–solvent interaction in different solvents. It would depend on the electron-donating capacity of RF molecule in organic solvents. The values of k_obs are inversely proportional to the viscosity of the medium as a result of diffusion-controlled processes.KEY WORDS: dielectric constant, kinetics, photolysis, riboflavin, solvent effect, viscosity     

Microsecond Time-Resolved Absorption Spectroscopy Used to Study CO Compounds of Cytochrome bd from Escherichia coli

Cytochrome bd is a tri-heme (b ₅₅₈, b ₅₉₅, d) respiratory oxygen reductase that is found in many bacteria including pathogenic species. It couples the electron transfer from quinol to O₂ with generation of an electrochemical proton gradient. We examined photolysis and subsequent recombination of CO with isolated cytochrome bd from Escherichia coli in one-electron reduced (MV) and fully reduced (R) states by microsecond time-resolved absorption spectroscopy at 532-nm excitation. Both Soret and visible band regions were examined. CO photodissociation from MV enzyme possibly causes fast (τ<1.5 µs) electron transfer from heme d to heme b ₅₉₅ in a small fraction of the protein, not reported earlier. Then the electron migrates to heme b ₅₅₈ (τ∼16 µs). It returns from the b-hemes to heme d with τ∼180 µs. Unlike cytochrome bd in the R state, in MV enzyme the apparent contribution of absorbance changes associated with CO dissociation from heme d is small, if any. Photodissociation of CO from heme d in MV enzyme is suggested to be accompanied by the binding of an internal ligand (L) at the opposite side of the heme. CO recombines with heme d (τ∼16 µs) yielding a transient hexacoordinate state (CO-Fe²⁺-L). Then the ligand slowly (τ∼30 ms) dissociates from heme d. Recombination of CO with a reduced heme b in a fraction of the MV sample may also contribute to the 30-ms phase. In R enzyme, CO recombines to heme d (τ∼20 µs), some heme b ₅₅₈ (τ∼0.2–3 ms), and finally migrates from heme d to heme b ₅₉₅ (τ∼24 ms) in ∼5% of the enzyme population. Data are consistent with the recent nanosecond study of Rappaport et al. conducted on the membranes at 640-nm excitation but limited to the Soret band. The additional phases were revealed due to differences in excitation and other experimental conditions.     

Purification of Two Nitrate Reductases from Xanthomonas maltophilia Grown in Aerobic Cultures

Xanthomonas maltophilia ATCC 17666 is an obligate aerobe that accumulates nitrite when grown on nitrate. Spectra of membranes from nitrate-grown cells exhibited b-type cytochrome peaks and A_615-630 indicative of d-type cytochrome but no absorption peaks corresponding to c-type cytochromes. The nitrate reductase (NR) activity was located in the membrane fraction. Triton X-100-extracted reduced methyl viologen-NRs were purified on DE-52, hydroxylapatite, and Sephacryl S-300 columns to specific activities of 52 to 67 μmol of nitrite formed per min per mg of protein. The cytochrome-containing NR_I separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis into a 135-kDa α-subunit, a 64-kDa β-subunit, and a 23-kDa γ-subunit with relative band intensities indicative of a 1:1:1 α/β/γ subunit ratio and a M_r of 222,000. The electronic spectrum of dithionite-reduced purified NR displayed peaks at 425, 528, and 558 nm, indicative of the presence of a cytochrome b, an interpretation consistent with the pyridine hemochrome spectrum formed. The cytochrome b of the NR was reduced under anaerobic conditions by menadiol and oxidized by nitrate with the production of nitrite. This NR contained 0.96 Mo, 12.5 nonheme iron, and 1 heme per 222 kDa: molybdopterin was detected with the Neurospora crassa nit-1 assay. A smaller reduced methyl viologen-NR (169 kDa), present in various concentrations in the Triton X-100 preparations, lacked a cytochrome spectrum and did not oxidize menadiol. The characteristics of the NRs and the absence of c-type cytochromes provide insights into why X. maltophilia accumulates nitrite.     

Thermal niche for in situ seed germination by Mediterranean mountain streams: model prediction and validation for Rhamnus persicifolia seeds

Background and Aims

Mediterranean mountain species face exacting ecological conditions of rainy, cold winters and arid, hot summers, which affect seed germination phenology. In this study, a soil heat sum model was used to predict field emergence of Rhamnus persicifolia, an endemic tree species living at the edge of mountain streams of central eastern Sardinia.

Methods

Seeds were incubated in the light at a range of temperatures (10–25 and 25/10 °C) after different periods (up to 3 months) of cold stratification at 5 °C. Base temperatures (T_b), and thermal times for 50 % germination (θ₅₀) were calculated. Seeds were also buried in the soil in two natural populations (Rio Correboi and Rio Olai), both underneath and outside the tree canopy, and exhumed at regular intervals. Soil temperatures were recorded using data loggers and soil heat sum (°Cd) was calculated on the basis of the estimated T_b and soil temperatures.

Key Results

Cold stratification released physiological dormancy (PD), increasing final germination and widening the range of germination temperatures, indicative of a Type 2 non-deep PD. T_b was reduced from 10·5 °C for non-stratified seeds to 2·7 °C for seeds cold stratified for 3 months. The best thermal time model was obtained by fitting probit germination against log °Cd. θ₅₀ was 2·6 log °Cd for untreated seeds and 2·17–2·19 log °Cd for stratified seeds. When θ₅₀ values were integrated with soil heat sum estimates, field emergence was predicted from March to April and confirmed through field observations.

Conclusions

T_b and θ₅₀ values facilitated model development of the thermal niche for in situ germination of R. persicifolia. These experimental approaches may be applied to model the natural regeneration patterns of other species growing on Mediterranean mountain waterways and of physiologically dormant species, with overwintering cold stratification requirement and spring germination.     

Activation of αVβ3 on Vascular Cells Controls Recognition of Prothrombin

Regulation of vascular homeostasis depends upon collaboration between cells of the vessel wall and blood coagulation system. A direct interaction between integrin α_Vβ₃ on endothelial cells and smooth muscle cells and prothrombin, the pivotal proenzyme of the blood coagulation system, is demonstrated and activation of the integrin is required for receptor engagement. Evidence that prothrombin is a ligand for α_Vβ₃ on these cells include: (a) prothrombin binds to purified α_Vβ₃ via a RGD recognition specificity; (b) prothrombin supports α_Vβ₃-mediated adhesion of stimulated endothelial cells and smooth muscle cells; and (c) endothelial cells, either in suspension and in a monolayer, recognize soluble prothrombin via α_Vβ₃. α_Vβ₃-mediated cell adhesion to prothrombin, but not to fibrinogen, required activation of the receptor. Thus, the functionality of the α_Vβ₃ receptor is ligand defined, and prothrombin and fibrinogen represent activation- dependent and activation-independent ligands. Activation of α_Vβ₃ could be induced not only by model agonists, PMA and Mn²⁺, but also by a physiologically relevant agonist, ADP. Inhibition of protein kinase C and calpain prevented activation of α_Vβ₃ on vascular cells, suggesting that these molecules are involved in the inside-out signaling events that activate the integrin. The capacity of α_Vβ₃ to interact with prothrombin may play a significant role in the maintenance of hemostasis; and, at a general level, ligand selection by α_Vβ₃ may be controlled by the activation state of this integrin.     

Cross-species comparison of site-specific evolutionary-rate variation in influenza haemagglutinin

We investigate the causes of site-specific evolutionary-rate variation in influenza haemagglutinin (HA) between human and avian influenza, for subtypes H1, H3, and H5. By calculating the evolutionary-rate ratio, ω = dN/dS as a function of a residue''s solvent accessibility in the three-dimensional protein structure, we show that solvent accessibility has a significant but relatively modest effect on site-specific rate variation. By comparing rates within HA subtypes among host species, we derive an upper limit to the amount of variation that can be explained by structural constraints of any kind. Protein structure explains only 20–40% of the variation in ω. Finally, by comparing ω at sites near the sialic-acid-binding region to ω at other sites, we show that ω near the sialic-acid-binding region is significantly elevated in both human and avian influenza, with the exception of avian H5. We conclude that protein structure, HA subtype, and host biology all impose distinct selection pressures on sites in influenza HA.     

Dynamic Light-Scattering Evidence for the Flexibility of Native Muscle Thin Filaments

We have obtained clear evidence for the flexibility of native scallop adductor thin filaments by studying the temperature and ionic strength dependence of the average decay constants obtained from intensity fluctuation spectroscopic (IFS) measurements. The low-angle (10-25°), average decay constants obtained from time autocorrelation functions of scattered light were independent of concentration (0.08-1.3 mg/ml), scaled with the ratio of temperature to solvent viscosity, T/η, over a range of 4-45°C, and yielded a value for the translational diffusion coefficient of D_T^5°C = (1.24 ± 0.06) × 10^-8 cm²/s. From this value and the Broersma relation for rigid rods, we find an average filament length of 1.06 ± 0.06 μm. Quantitative sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that at high temperatures (> 35°C) or in 0.6 M NaCl, tropomyosin completely dissociates from native thin filaments. Decay constants from high-angle (60-150°C) IFS temperature dependence measurements do not scale with T/η and hence do not show the temperature dependence expected for rigid rods. The differences are not due to any change in length distribution of filaments with temperature or to the free tropomyosin in solution, but are attributed to nonrigid motions of the filaments. Similar experiments on samples in high- and low-salt solvents gave results consistent with this interpretation.     

Phosphorylation of cMyBP-C Affects Contractile Mechanisms in a Site-specific Manner

Cardiac myosin binding protein-C (cMyBP-C) is a cardiac-specific, thick-filament regulatory protein that is differentially phosphorylated at Ser²⁷³, Ser²⁸², and Ser³⁰² by various kinases and modulates contraction. In this study, phosphorylation-site-specific effects of cMyBP-C on myocardial contractility and cross-bridge kinetics were studied by sinusoidal analysis in papillary and trabecular muscle fibers isolated from t/t (cMyBP-C-null) mice and in their counterparts in which cMyBP-C contains the ADA (Ala²⁷³-Asp²⁸²-Ala³⁰²), DAD (Asp²⁷³-Ala²⁸²-Asp³⁰²), and SAS (Ser²⁷³-Ala²⁸²-Ser³⁰²) mutations; the results were compared to those from mice expressing the wild-type (WT) transgene on the t/t background. Under standard activating conditions, DAD fibers showed significant decreases in tension (∼50%), stiffness, the fast apparent rate constant 2πc, and its magnitude C, as well as its magnitude H, but an increase in the medium rate constant 2πb, with respect to WT. The t/t fibers showed a smaller drop in stiffness and a significant decrease in 2πc that can be explained by isoform shift of myosin heavy chain. In the pCa-tension study using the 8 mM phosphate (Pi) solution, there was hardly any difference in Ca²⁺ sensitivity (pCa₅₀) and cooperativity (n_H) between the mutant and WT samples. However, in the solutions without Pi, DAD showed increased n_H and slightly decreased pCa₅₀. We infer from these observations that the nonphosphorylatable residue 282 combined with phosphomimetic residues Asp²⁷³ and/or Asp³⁰² (in DAD) is detrimental to cardiomyocytes by lowering isometric tension and altering cross-bridge kinetics with decreased 2πc and increased 2πb. In contrast, a single change of residue 282 to nonphosphorylatable Ala (SAS), or to phosphomimetic Asps together with the changes of residues 273 and 302 to nonphosphorylatable Ala (ADA) causes minute changes in fiber mechanics.     

How DASPMI reveals mitochondrial membrane potential: fluorescence decay kinetics and steady-state anisotropy in living cells

Spectroscopic responses of the potentiometric probe 2-(4-(dimethylamino)styryl)-1-methylpyridinium iodide (DASPMI) were investigated in living cells by means of a time- and space-correlated single photon counting technique. Spatially resolved fluorescence decays from single mitochondria or only a very few organelles of XTH2 cells exhibited three-exponential decay kinetics. Based on DASPMI photophysics in a variety of solvents, these lifetimes were attributed to the fluorescence from the locally excited state, intramolecular charge transfer state, and twisted intramolecular charge transfer state. A considerable variation in lifetimes among mitochondria of different morphologies and within single cells was evident, corresponding to high physiological variations within single cells. Considerable shortening of the short lifetime component (τ₁) under a high-membrane-potential condition, such as in the presence of ATP and/or substrate, was similar to quenching and a dramatic decrease of lifetime in polar solvents. Under these conditions τ₂ and τ₃ increased with decreasing contribution. Inhibiting respiration by cyanide resulted in a notable increase in the mean lifetime and a decrease in mitochondrial fluorescence. Increased DASPMI fluorescence under conditions that elevate the mitochondrial membrane potential has been attributed to uptake according to Nernst distributions, delocalization of π-electrons, quenching processes of the methyl pyridinium moiety, and restricted torsional dynamics at the mitochondrial inner membrane. Accordingly, determination of anisotropy in DASPMI-stained mitochondria in living cells revealed a dependence of anisotropy on the membrane potential. The direct influence of the local electric field on the transition dipole moment of the probe and its torsional dynamics monitor changes in mitochondrial energy status within living cells.     

Effect of Sarcoplasmic Reticulum (SR) Calcium Content on SR Calcium Release Elicited by Small Voltage-Clamp Depolarizations in Frog Cut Skeletal Muscle Fibers Equilibrated with 20 mM EGTA

Cut muscle fibers from Rana temporaria (sarcomere length, 3.5–3.9 μm; 14–16°C) were mounted in a double Vaseline-gap chamber and equilibrated with an external solution that contained tetraethyl ammonium– gluconate and an internal solution that contained Cs as the principal cation, 20 mM EGTA, and 0 Ca. Fibers were stimulated with a voltage-clamp pulse protocol that consisted of pulses to −70, −65, −60, −45, and −20 mV, each separated by 400-ms periods at −90 mV. The change in total Ca that entered into the myoplasm (Δ[Ca_T]) and the Ca content of the SR ([Ca_SR]) were estimated with the EGTA/phenol red method (Pape, P.C., D.-S. Jong, and W.K. Chandler. 1995. J. Gen. Physiol. 106:259–336). Fibers were stimulated with the pulse protocol, usually every 5 min, so that the resting value of [Ca_SR] decreased from its initial value of 1,700–2,300 μM to values near or below 100 μM after 18–30 stimulations. Three main findings for the voltage pulses to −70, −65, and −60 mV are: (a) the depletion-corrected rate of Ca release (release permeability) showed little change when [Ca_SR] decreased from its highest level (>1,700 μM) to ∼1,000 μM; (b) as [Ca_SR] decreased below 1,000 μM, the release permeability increased to a maximum level when [Ca_SR] was near 300 μM that was on average about sevenfold larger than the values observed for [Ca_SR] > 1,000 μM; and (c) as [Ca_SR] decreased from ∼300 μM to <100 μM, the release permeability decreased, reaching half its maximum value when [Ca_SR] was ∼110 μM on average. It was concluded that finding b was likely due to a decrease in Ca inactivation, while finding c was likely due to a decrease in Ca-induced Ca release.