A Thermodynamic Model of the Cardiac Sarcoplasmic/Endoplasmic Ca (SERCA) Pump

We present a biophysically based kinetic model of the cardiac SERCA pump that consolidates a range of experimental data into a consistent and thermodynamically constrained framework. The SERCA model consists of a number of sub-states with partial reactions that are sensitive to Ca²⁺ and pH, and to the metabolites MgATP, MgADP, and Pi. Optimization of model parameters to fit experimental data favors a fully cooperative Ca²⁺-binding mechanism and predicts a Ca²⁺/H⁺ counter-transport stoichiometry of 2. Moreover, the order of binding of the partial reactions, particularly the binding of MgATP, proves to be a strong determinant of the ability of the model to fit the data. A thermodynamic investigation of the model indicates that the binding of MgATP has a large inhibitory effect on the maximal reverse rate of the pump. The model is suitable for integrating into whole-cell models of cardiac electrophysiology and Ca²⁺ dynamics to simulate the effects on the cell of compromised metabolism arising in ischemia and hypoxia.     

Nonlinear force-length relationship in the ADP-induced contraction of skeletal myofibrils

The regulatory mechanism of sarcomeric activity has not been fully clarified yet because of its complex and cooperative nature, which involves both Ca²⁺ and cross-bridge binding to the thin filament. To reveal the mechanism of regulation mediated by the cross-bridges, separately from the effect of Ca²⁺, we investigated the force-sarcomere length (SL) relationship in rabbit skeletal myofibrils (a single myofibril or a thin bundle) at SL > 2.2 μm in the absence of Ca²⁺ at various levels of activation by exogenous MgADP (4-20 mM) in the presence of 1 mM MgATP. The individual SLs were measured by phase-contrast microscopy to confirm the homogeneity of the striation pattern of sarcomeres during activation. We found that at partial activation with 4-8 mM MgADP, the developed force nonlinearly depended on the length of overlap between the thick and the thin filaments; that is, contrary to the maximal activation, the maximal active force was generated at shorter overlap. Besides, the active force became larger, whereas this nonlinearity tended to weaken, with either an increase in [MgADP] or the lateral osmotic compression of the myofilament lattice induced by the addition of a macromolecular compound, dextran T-500. The model analysis, which takes into account the [MgADP]-and the lattice-spacing-dependent probability of cross-bridge formation, was successfully applied to account for the force-SL relationship observed at partial activation. These results strongly suggest that the cross-bridge works as a cooperative activator, the function of which is highly sensitive to as little as ≤1 nm changes in the lattice spacing.     

History-dependent properties of skeletal muscle myofibrils contracting along the ascending limb of the force–length relationship

There is a history dependence of skeletal muscle contraction: stretching activated muscles induces a long-lasting force enhancement, while shortening activated muscles induces a long-lasting force depression. These history-dependent properties cannot be explained by the current model of muscle contraction, and its mechanism is unknown. The purposes of this study were (i) to evaluate if force enhancement and force depression are present at short lengths (the ascending limb of the force–length (FL) relationship), (ii) to evaluate if the history-dependent properties are associated with sarcomere length (SL) non-uniformity and (iii) to determine the effects of cross-bridge (de)activation on force depression. Rabbit psoas myofibrils were isolated and attached between two microneedles for force measurements. Images of the myofibrils were projected onto a linear photodiode array for measurements of SL. Myofibrils were activated by either Ca²⁺ or MgADP; the latter induces cross-bridge attachment to actin independently of Ca²⁺. Activated myofibrils were subjected to three stretches or shortenings (approx. 4% SL at approx. 0.07 µm s⁻¹ sarcomere⁻¹) along the ascending limb of the FL relationship separated by periods (approx. 5 s) of isometric contraction. Force after stretch was higher than force after shortening at similar SLs. The differences in force could not be explained by SL non-uniformity. The FL relationship produced by Ca²⁺- and MgADP-activated myofibrils were similar in stretch experiments, but after shortening MgADP activation produced forces that were higher than Ca²⁺ activation. Since MgADP induces the formation of strongly bound cross-bridges, this result suggests that force depression following shortening is associated with cross-bridge deactivation.     

Cooperative Cross-Bridge Activation of Thin Filaments Contributes to the Frank-Starling Mechanism in Cardiac Muscle

Myosin cross-bridges play an important role in the regulation of thin-filament activation in cardiac muscle. To test the hypothesis that sarcomere length (SL) modulation of thin-filament activation by strong-binding cross-bridges underlies the Frank-Starling mechanism, we inhibited force and strong cross-bridge binding to intermediate levels with sodium vanadate (Vi). Force and stiffness varied proportionately with [Ca²⁺] and [Vi]. Increasing [Vi] (decreased force) reduced the pCa₅₀ of force-[Ca²⁺] relations at 2.3 and 2.0 μm SL, with little effect on slope (n_H). When maximum force was inhibited to ∼40%, the effects of SL on force were diminished at lower [Ca²⁺], whereas at higher [Ca²⁺] (pCa < 5.6) the relative influence of SL on force increased. In contrast, force inhibition to ∼20% significantly reduced the sensitivity of force-[Ca²⁺] relations to changes in both SL and myofilament lattice spacing. Strong cross-bridge binding cooperatively induced changes in cardiac troponin C structure, as measured by dichroism of 5′ iodoacetamido-tetramethylrhodamine-labeled cardiac troponin C. This apparent cooperativity was reduced at shorter SL. These data emphasize that SL and/or myofilament lattice spacing modulation of the cross-bridge component of cardiac thin-filament activation contributes to the Frank-Starling mechanism.     

Cross-bridge scheme and force per cross-bridge state in skinned rabbit psoas muscle fibers.

The rate and association constants (kinetic constants) which comprise a seven state cross-bridge scheme were deduced by sinusoidal analysis in chemically skinned rabbit psoas muscle fibers at 20 degrees C, 200 mM ionic strength, and during maximal Ca2+ activation (pCa 4.54-4.82). The kinetic constants were then used to calculate the steady state probability of cross-bridges in each state as the function of MgATP, MgADP, and phosphate (Pi) concentrations. This calculation showed that 72% of available cross-bridges were (strongly) attached during our control activation (5 mM MgATP, 8 mM Pi), which agreed approximately with the stiffness ratio (active:rigor, 69 +/- 3%); active stiffness was measured during the control activation, and rigor stiffness after an induction of the rigor state. By assuming that isometric tension is a linear combination of probabilities of cross-bridges in each state, and by measuring tension as the function of MgATP, MgADP, and Pi concentrations, we deduced the force associated with each cross-bridge state. Data from the osmotic compression of muscle fibers by dextran T500 were used to deduce the force associated with one of the cross-bridge states. Our results show that force is highest in the AM*ADP.Pi state (A = actin, M = myosin). Since the state which leads into the AM*ADP.Pi state is the weakly attached AM.ADP.Pi state, we confirm that the force development occurs on Pi isomerization (AM.ADP.Pi --> AM*ADP.Pi). Our results also show that a minimal force change occurs with the release of Pi or MgADP, and that force declines gradually with ADP isomerization (AM*ADP -->AM.ADP), ATP isomerization (AM+ATP-->AM*ATP), and with cross-bridge detachment. Force of the AM state agreed well with force measured after induction of the rigor state, indicating that the AM state is a close approximation of the rigor state. The stiffness results obtained as functions of MgATP, MgADP, and Pi concentrations were generally consistent with the cross-bridge scheme.     

Cooperative interactions between calcium-binding sites on glycerinated muscle fibers the influence of cross-bridge attachment

A double isotope technique and EGTA buffers were used to measure the binding of Ca²⁺ to rabbit psoas muscle fibers extracted with detergent and glycerol. These experiments were designed to test the effect of rigor complex formation, determined by the degree of filament overlap, on the properties of the Ca²⁺-binding sites in the intact filament lattice. In the presence of 5 mM MgCl₂ (no ATP), reduction of filament overlap was associated with a reduced binding of Ca²⁺ over the entire range of free Ca²⁺ concentrations (5 · 10^?8 – 2 · 10^?5 M). With maximum filament overlap (sarcomere length 2.1–2.2 μm) the maximum bound Ca²⁺ was equivalent to 4 mol Ca²⁺/mol troponin and there was significant positive interaction between binding sites, as shown by Scatchard and Hill plots. With no filament overlap (sarcomere length 3.8–4.4 μm) the maximum bound Ca²⁺ was equivalent to 3 μmol Ca²⁺/mol troponin and graphical analysis indicated a single class of non-interacting sites. The data provide evidence that when cross-bridge attachments between actin and myosin filaments are formed not only does an additional Ca²⁺ binding site appear, but cooperative properties are imposed upon the binding sites.     

Regulatory mechanism of length-dependent activation in skinned porcine ventricular muscle: role of thin filament cooperative activation in the Frank-Starling relation

Cardiac sarcomeres produce greater active force in response to stretch, forming the basis of the Frank-Starling mechanism of the heart. The purpose of this study was to provide the systematic understanding of length-dependent activation by investigating experimentally and mathematically how the thin filament “on–off” switching mechanism is involved in its regulation. Porcine left ventricular muscles were skinned, and force measurements were performed at short (1.9 µm) and long (2.3 µm) sarcomere lengths. We found that 3 mM MgADP increased Ca²⁺ sensitivity of force and the rate of rise of active force, consistent with the increase in thin filament cooperative activation. MgADP attenuated length-dependent activation with and without thin filament reconstitution with the fast skeletal troponin complex (sTn). Conversely, 20 mM of inorganic phosphate (Pi) decreased Ca²⁺ sensitivity of force and the rate of rise of active force, consistent with the decrease in thin filament cooperative activation. Pi enhanced length-dependent activation with and without sTn reconstitution. Linear regression analysis revealed that the magnitude of length-dependent activation was inversely correlated with the rate of rise of active force. These results were quantitatively simulated by a model that incorporates the Ca²⁺-dependent on–off switching of the thin filament state and interfilament lattice spacing modulation. Our model analysis revealed that the cooperativity of the thin filament on–off switching, but not the Ca²⁺-binding ability, determines the magnitude of the Frank-Starling effect. These findings demonstrate that the Frank-Starling relation is strongly influenced by thin filament cooperative activation.     

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.     

The role of thin filament cooperativity in cardiac length-dependent calcium activation

Length-dependent activation (LDA) is a prominent feature of cardiac muscle characterized by decreases in the Ca²⁺ levels required to generate force (i.e., increases in Ca²⁺ sensitivity) when muscle is stretched. Previous studies have concluded that LDA originates from the increased ability of (strong) cross-bridges to attach when muscle is lengthened, which in turn enhances Ca²⁺ binding to the troponin C (TnC) subunit of the troponin complex. However, our results demonstrate that inhibition of strong cross-bridge attachment with blebbistatin had no effect on the length-dependent modulation of Ca²⁺ sensitivity (i.e., EC₅₀) or Ca²⁺ cooperativity, suggesting that LDA originates upstream of cross-bridge attachment. To test whether LDA arises from length dependence of thin-filament activation, we replaced native cTnC with a mutant cTnC (DM-TnC) that is incapable of binding Ca²⁺. Although progressive replacement of native cTnC with DM-TnC caused an expected monotonic decrease in the maximal force (F_max), DM-TnC incorporation induced much larger increases in EC₅₀ and decreases in Ca²⁺ cooperativity at short lengths than at long lengths. These findings support the conclusion that LDA arises primarily from the influence of length on the modulation of the Ca²⁺ cooperativity arising from interaction between adjacent troponin-tropomyosin complexes on the thin filament.     

The Effects of Ca2+ and MgADP on Force Development during and after Muscle Length Changes

The goal of this study was to compare the effects of Ca²⁺ and MgADP activation on force development in skeletal muscles during and after imposed length changes. Single fibres dissected from the rabbit psoas were (i) activated in pCa²⁺4.5 and pCa²⁺6.0, or (ii) activated in pCa²⁺4.5 before and after administration of 10 mM MgADP. Fibres were activated in sarcomere lengths (SL) of 2.65 µm and 2.95 µm, and subsequently stretched or shortened (5%SL at 1.0 SL.s⁻¹) to reach a final SL of 2.80 µm. The kinetics of force during stretch were not altered by pCa²⁺ or MgADP, but the fast change in the slope of force development (P₁) observed during shortening and the corresponding SL extension required to reach the change (L₁) were higher in pCa²⁺6.0 (P₁ = 0.22±0.02 P_o; L₁ = 5.26±0.24 nm.HS^.1) than in pCa²⁺4.5 (P₁ = 0.15±0.01 P_o; L₁ = 4.48±0.25 nm.HS^.1). L₁ was also increased by MgADP activation during shortening. Force enhancement after stretch was lower in pCa²⁺4.5 (14.9±5.4%) than in pCa²⁺6.0 (38.8±7.5%), while force depression after shortening was similar in both Ca²⁺ concentrations. The stiffness accompanied the force behavior after length changes in all situations. MgADP did not affect the force behavior after length changes, and stiffness did not accompany the changes in force development after stretch. Altogether, these results suggest that the mechanisms of force generation during and after stretch are different from those obtained during and after shortening.     

Dynamic coupling of regulated binding sites and cycling myosin heads in striated muscle

In an activated muscle, binding sites on the thin filament and myosin heads switch frequently between different states. Because the status of the binding sites influences the status of the heads, and vice versa, the binding sites and myosin heads are dynamically coupled. The functional consequences of this coupling were investigated using MyoSim, a new computer model of muscle. MyoSim extends existing models based on Huxley-type distribution techniques by incorporating Ca²⁺ activation and cooperative effects. It can also simulate arbitrary cross-bridge schemes set by the researcher. Initial calculations investigated the effects of altering the relative speeds of binding-site and cross-bridge kinetics, and of manipulating cooperative processes. Subsequent tests fitted simulated force records to experimental data recorded using permeabilized myocardial preparations. These calculations suggest that the rate of force development at maximum activation is limited by myosin cycling kinetics, whereas the rate at lower levels of activation is limited by how quickly binding sites become available. Additional tests investigated the behavior of transiently activated cells by driving simulations with experimentally recorded Ca²⁺ signals. The unloaded shortening profile of a twitching myocyte could be reproduced using a model with two myosin states, cooperative activation, and strain-dependent kinetics. Collectively, these results demonstrate that dynamic coupling of binding sites and myosin heads is important for contractile function.     

Omecamtiv Mecarbil Slows Myosin Kinetics in Skinned Rat Myocardium at Physiological Temperature

Heart failure is a life-threatening condition that occurs when the heart muscle becomes weakened and cannot adequately circulate blood and nutrients around the body. Omecamtiv mecarbil (OM) is a compound that has been developed to treat systolic heart failure via targeting the cardiac myosin heavy chain to increase myocardial contractility. Biophysical and biochemical studies have found that OM increases calcium (Ca²⁺) sensitivity of contraction by prolonging the myosin working stroke and increasing the actin-myosin cross-bridge duty ratio. Most in vitro studies probing the effects of OM on cross-bridge kinetics and muscle force production have been conducted at subphysiological temperature, even though temperature plays a critical role in enzyme activity and cross-bridge function. Herein, we used skinned, ventricular papillary muscle strips from rats to investigate the effects of [OM] on Ca²⁺-activated force production, cross-bridge kinetics, and myocardial viscoelasticity at physiological temperature (37°C). We find that OM only increases myocardial contractility at submaximal Ca²⁺ activation levels and not maximal Ca²⁺ activation levels. As [OM] increased, the kinetic rate constants for cross-bridge recruitment and detachment slowed for both submaximal and maximal Ca²⁺-activated conditions. These findings support a mechanism by which OM increases cardiac contractility at physiological temperature via increasing cross-bridge contributions to thin-filament activation as cross-bridge kinetics slow and the duration of cross-bridge attachment increases. Thus, force only increases at submaximal Ca²⁺ activation due to cooperative recruitment of neighboring cross-bridges, because thin-filament activation is not already saturated. In contrast, OM does not increase myocardial force production for maximal Ca²⁺-activated conditions at physiological temperature because cooperative activation of thin filaments may already be saturated.     

Kinetic and thermodynamic studies of the cross-bridge cycle in rabbit psoas muscle fibers.

The effect of temperature on elementary steps of the cross-bridge cycle was investigated with sinusoidal analysis technique in skinned rabbit psoas fibers. We studied the effect of MgATP on exponential process (C) to characterize the MgATP binding step and cross-bridge detachment step at six different temperatures in the range 5-30 degrees C. Similarly, we studied the effect of MgADP on exponential process (C) to characterize the MgADP binding step. We also studied the effect of phosphate (Pi) on exponential process (B) to characterize the force generation step and Pi-release step. From the results of these studies, we deduced the temperature dependence of the kinetic constants of the elementary steps and their thermodynamic properties. We found that the MgADP association constant (K0) and the MgATP association constant (K1) significantly decreased when the temperature was increased from 5 to 20 degrees C, implying that nucleotide binding became weaker at higher temperatures. K0 and K1 did not change much in the 20-30 degree C range. The association constant of Pi to cross-bridges (K5) did not change much with temperature. We found that Q10 for the cross-bridge detachment step (k2) was 2.6, and for its reversal step (k-2) was 3.0. We found that Q10 for the force generation step (Pi-isomerization step, k4) was 6.8, and its reversal step (k-4) was 1.6. The equilibrium constant of the detachment step (K2) was not affected much by temperature, whereas the equilibrium constant of the force generation step (K4) increased significantly with temperature increase. Thus, the force generation step consists of an endothermic reaction. The rate constant of the rate-limiting step (k6) did not change much with temperature, whereas the ATP hydrolysis rate increased significantly with temperature increase. We found that the force generation step accompanies a large entropy increase and a small free energy change; hence, this step is an entropy-driven reaction. These observations are consistent with the hypothesis that the hydrophobic interaction between residues of actin and myosin underlies the mechanism of force generation. We conclude that the force generation step is the most temperature-sensitive step among elementary steps of the cross-bridge cycle, which explains increased isometric tension at high temperatures in rabbit psoas fibers.     

Cardiac mechanical efficiency is preserved in primary cardiac hypertrophy despite impaired mechanical function

Increased heart size is a major risk factor for heart failure and premature mortality. Although abnormal heart growth subsequent to hypertension often accompanies disturbances in mechano-energetics and cardiac efficiency, it remains uncertain whether hypertrophy is their primary driver. In this study, we aimed to investigate the direct association between cardiac hypertrophy and cardiac mechano-energetics using isolated left-ventricular trabeculae from a rat model of primary cardiac hypertrophy and its control. We evaluated energy expenditure (heat output) and mechanical performance (force length work production) simultaneously at a range of preloads and afterloads in a microcalorimeter, we determined energy expenditure related to cross-bridge cycling and Ca²⁺ cycling (activation heat), and we quantified energy efficiency. Rats with cardiac hypertrophy exhibited increased cardiomyocyte length and width. Their trabeculae showed mechanical impairment, evidenced by lower force production, extent and kinetics of shortening, and work output. Lower force was associated with lower energy expenditure related to Ca²⁺ cycling and to cross-bridge cycling. However, despite these changes, both mechanical and cross-bridge energy efficiency were unchanged. Our results show that cardiac hypertrophy is associated with impaired contractile performance and with preservation of energy efficiency. These findings provide direction for future investigations targeting metabolic and Ca²⁺ disturbances underlying cardiac mechanical and energetic impairment in primary cardiac hypertrophy.     

A Mathematical Model of Action Potential in Cells of Vascular Plants

A mathematical model of action potential (AP) in vascular plants cells has been worked out. The model takes into account actions of plasmalemma ion transport systems (K⁺, Cl^? and Ca²⁺ channels; H⁺- and Ca²⁺-ATPases; 2H⁺/Cl^? symporter; and H⁺/K⁺ antiporter), changes of ion concentrations in the cell and in the extracellular space, cytoplasmic and apoplastic buffer capacities and the temperature dependence of active transport systems. The model of AP simulates a stationary level of the membrane potential and ion concentrations, generation of AP induced by electrical stimulation and gradual cooling and the impact of external Ca²⁺ for AP development. The model supports a hypothesis about participation of H⁺-ATPase in AP generation.     

Sarcomere lattice geometry influences cooperative myosin binding in muscle

In muscle, force emerges from myosin binding with actin (forming a cross-bridge). This actomyosin binding depends upon myofilament geometry, kinetics of thin-filament Ca²⁺ activation, and kinetics of cross-bridge cycling. Binding occurs within a compliant network of protein filaments where there is mechanical coupling between myosins along the thick-filament backbone and between actin monomers along the thin filament. Such mechanical coupling precludes using ordinary differential equation models when examining the effects of lattice geometry, kinetics, or compliance on force production. This study uses two stochastically driven, spatially explicit models to predict levels of cross-bridge binding, force, thin-filament Ca²⁺ activation, and ATP utilization. One model incorporates the 2-to-1 ratio of thin to thick filaments of vertebrate striated muscle (multi-filament model), while the other comprises only one thick and one thin filament (two-filament model). Simulations comparing these models show that the multi-filament predictions of force, fractional cross-bridge binding, and cross-bridge turnover are more consistent with published experimental values. Furthermore, the values predicted by the multi-filament model are greater than those values predicted by the two-filament model. These increases are larger than the relative increase of potential inter-filament interactions in the multi-filament model versus the two-filament model. This amplification of coordinated cross-bridge binding and cycling indicates a mechanism of cooperativity that depends on sarcomere lattice geometry, specifically the ratio and arrangement of myofilaments.     

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.     

Effects of monovalent cations on Ca uptake by skeletal and cardiac muscle sarcoplasmic reticulum

Ca²⁺ transport by the sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase (SERCA) is sensitive to monovalent cations. Possible K⁺ binding sites have been identified in both the cytoplasmic P-domain and the transmembrane transport-domain of the protein. We measured Ca²⁺ transport into SR vesicles and SERCA ATPase activity in the presence of different monovalent cations. We found that the effects of monovalent cations on Ca²⁺ transport correlated in most cases with their direct effects on SERCA. Choline⁺, however, inhibited uptake to a greater extent than could be accounted for by its direct effect on SERCA suggesting a possible effect of choline on compensatory charge movement during Ca²⁺ transport. Of the monovalent cations tested, only Cs⁺ significantly affected the Hill coefficient of Ca²⁺ transport (n_H). An increase in n_H from ∼2 in K⁺ to ∼3 in Cs⁺ was seen in all of the forms of SERCA examined. The effects of Cs⁺ on the maximum velocity of Ca²⁺ uptake were also different for different forms of SERCA but these differences could not be attributed to differences in the putative K⁺ binding sites of the different forms of the protein.     

Cytosolic Ca2+ and H+ buffers in green algae: A comment

Summary Recently Plieth et al. [Protoplasma (1997) 198: 107–124; 199: 223] gave a quantitative picture of the Ca²⁺ and H⁺ buffers in green algae which we would like to comment. In that paper a mechanistic model was derived which describes the relationship between cytosolic Ca²⁺ and H⁺ assuming that Ca²⁺ and H⁺ interact with the same binding site of a Ca²⁺-H⁺-exchange buffer. But the increase of the cytosolic free Ca²⁺ concentration observed upon acidification can alternatively be described by a co-operative (n=2) protonation of a Ca²⁺/H⁺-binding buffer pointing to an allosteric mechanism of Ca²⁺ liberation. Furthermore we present evidences that the cytosolic buffer capacities for H⁺ (90 mM/pH) and Ca²⁺ (20 mM/pCa) given for Eremosphaera viridis were overestimated by a factor of three and three orders of magnitude, respectively.Abbreviations [Ca²⁺]_c free cytosolic - Ca²⁺ concentration     

Reactions of corn root tissue to calcium

Washing corn (Zea mays L.) root tissue in water causes loss of about one-third of the exchangeable Ca²⁺ over the first 10 to 15 minutes. Upon transfer to K⁺-containing solutions, the tissue shows a short period of rapid K⁺ influx which subsequently declines. Addition of 0.1 millimolar Ca²⁺ decreases the initial rapid K⁺ influx, but increases the sustained rate of K⁺ and Cl⁻ uptake. It was confirmed (Elzam and Hodges 1967 Plant Physiol 42: 1483-1488) that 0.1 millimolar Ca²⁺ is more effective than higher concentrations for the initial inhibition, and that Mg²⁺ will substitute.

The inhibition arises from a mild shock affect of restoring Ca²⁺. With 0.1 millimolar Ca²⁺ net H⁺ efflux is blocked for 10 to 15 minutes and the cells are depolarized by about 30 millivolts. However, 1 millimolar Ca²⁺ rapidly produces increased K⁺ influx and blocks net H⁺ efflux for only a few minutes; blockage is preceded by a brief net H⁺ influx which may restore and increase ion transport by reactivating the plasmalemma H⁺-ATPase.

Stimulation of electrogenic H⁺-pumping with fusicoccin eliminates the shock responses and minimizes Ca²⁺ effects on K⁺ influx. Fusicoccin also strongly decreases Ca²⁺ influx, but has no effect on Ca²⁺ efflux. Ice temperatures and high pH decreased Ca²⁺ efflux, but uncoupler and chlorpromazine did not.

It is suggested that the inhibitory and promotive actions of Ca²⁺ are manifested through decreases or increases in the protonmotive force.