Effect of the membrane potential on the Mg2+,ATP-dependent transport of Ca2+ across smooth muscle sarcolemma

The effect of the membrane potential (K(+)-valinomycin system) on the Mg2+, ATP-dependent transport of Ca2+ in inside-out vesicles of myometrium sarcolemma has been studied. The membrane potential was identified by using a cyanine potential-sensitive probe, diS-C3-(5). In the presence of valinomycin (5.10(-8) M) the inside-out directed K+ gradient (delta psi = -86 mV, with a negative charge inside) stimulated the initial rate of the energy-dependent accumulation of Ca2+ transfer whereas the oppositely directed K+ gradient (delta psi = +72 mV, with a positive charge inside) had no effect on this process. The K+ gradient was formed by isotonic substitution of K+ in intra- or extravesicular space for choline +. At the same time, in the absence of K+ gradient the Mg2+, ATP-dependent accumulation of Ca2+ in membrane vesicles did not depend on the chemical nature of the cations (K+ or choline+) used for isotonicity. The decrease of delta psi from 0 to -86 mV affects the initial rate of Ca2+ accumulation but not the maximal content of the accumulated cation. Preliminary dissipation of the membrane potential (delta psi = -86 mV) in Mg2(+)-free isotonic (with respect of K+ and choline+) media containing ATP and Ca2+ resulted in the inhibition of Mg2+, ATP-dependent Ca2+ transport induced by subsequent addition of Mg2+. These results indicate that the negative (intravesicular) electrical potential activates the Ca-pump of smooth muscle sarcolemma. This activation is based on the increase in the turnover number of the Ca2+ transporting system but not on its affinity for the transfer substrate. The use of the absolute reaction rates theory made it possible to establish that the Ca-pump effectuates the transport of a single positive charge in inside-out vesicles of smooth muscle plasma membranes, i.e., the energy-dependent transport of Ca2+ occurs either as a symport (with an anion (Cl-) or an antiport with a monovalent cation (K+) or a proton. It is assumed that the potential dependence of the Ca-pump in the smooth muscle plasma membrane plays a role in the realization of effects of mediators and physiologically active substances that are manifested as stimulation of the contractile response and depolarization of the sarcolemma. In is quite probable that the delta psi-dependent Ca-pump is also responsible for the maintenance of intracellular homeostasis of monovalent cations (K+, H+, Cl-) in smooth muscle tissues.     

cAMP-dependent phosphorylation inhibits potential-dependent passive transport of Ca2+ in myometrial sarcolemma vesicles

It is established that Ca2+ transport from the predominantly inverted vesicles of pig myometrium sarcolemma depends on the value of the membrane potential which is created on vesicles by the K+-valinomycin system. It is shown that variations in the membrane potential from -60 to +30 mV cause acceleration of the calcium transport from the vesicles, the maximal transport being observed at delta psi from 0 up to +30 mV. The endogenic and exogenic cAMP-dependent phosphorylation of plasma membrane proteins inhibits the passive transport of calcium at all the membrane potential values studied. A degree of potential-dependent Ca2+ transport inhibition correlates with the value of cAMP-dependent phosphorylation of sarcolemma proteins.     

Passive transport of calcium into plasma membrane fractions of myometrium cells

The apparent values of intravesicular volume (45 microliter/mg of protein), maximal capacity of adsorbed calcium binding on the inner surface of the vesicles (4.5 nmol/mg of protein) and dissociation constants for the Ca2+-binding site complexes (36 microM) were determined from the analysis of peculiarities of passive transport of 45Ca2+ into cow myometrium sarcolemmal vesicles. The kinetics of passive efflux of ionized Ca2+ from the vesicles is described by a two-phase exponential curve. Dilution of the vesicles with a dilution medium is associated with a rapid efflux of ionized Ca2+ from the intravesicular space resulting in dissociation of the Ca2+-binding site complexes on the inner surface of the vesicles and, correspondingly, in the passage from a rapid to the slow phase of Ca2+ efflux from the vesicles which is limited by the dissociation of the Ca2+-binding site complexes. The values of the apparent rate constants for the transmembrane transfer of Ca2+ and dissociation of the Ca2+-binding site complexes (0.73 and 0.02 min-1, respectively) and the permeability of sarcolemmal vesicles for the cation (10(-15) mol of Ca2+/cm2.s) were determined. Alkalinization of the dilution medium stimulates 45Ca2+ release from the vesicles. The blockers of passive Co2+ and Mn2+ transport injected into the vesicles inhibit the efflux of 45Ca2+ from the vesicles. The data obtained were used to analyze the role of sarcolemma in the Ca2+ control of myometrium contraction.     

Distribution of electrical potential, pH, free Ca2+, and volume inside cultured adult rabbit cardiac myocytes during chemical hypoxia: a multiparameter digitized confocal microscopic study.

Exploiting the optical sectioning capabilities of laser scanning confocal microscopy and using parameter-specific fluorescent probes, we determined the distribution of pH, free Ca2+, electrical potential, and volume inside cultured adult rabbit cardiac myocytes during ATP depletion and reductive stress with cyanide and 2-deoxyglucose ("chemical hypoxia"). During normoxic incubations, myocytes exhibited a cytosolic pH of 7.1 and a mitochondrial pH of 8.0 (delta pH = 0.9 units). Sarcolemmal membrane potential (delta psi) was -80 mV, and mitochondrial delta psi was as high as -100 mV, yielding a mitochondrial protonmotive force (delta p) of -155 mV (delta P = delta psi - 60 delta pH). After 30 min of chemical hypoxia, mitochondrial delta pH decreased to 0.5 pH units, but mitochondrial delta psi remained essentially unchanged. By 40 min, delta pH was collapsed, and mitochondrial and cytosolic free Ca2+ began to increase. Mitochondrial and sarcolemmal delta psi remained high. as Ca2+ rose, myocytes shortened, hypercontracted, and blebbed with a 30% decrease of cell volume. After hypercontraction, extensive mitochondrial Ca2+ loading occurred. After another few minutes, mitochondrial depolarized completely and released their load of Ca2+. After many more minutes, the sarcolemmal permeability barrier broke down, and viability was lost. These studies demonstrate a sequence of subcellular ionic and electrical changes that may underlie the progression to irreversible hypoxic injury.     

Regulation by Ca2+-calmodulin-dependent phosphorylation of passive transport of Ca2+ in the myometrial sarcolemma

Closed vesiculate preparations of pig myometrium sarcolemma (predominantly with inside-out orientation) are characterized by passive permeability for Ca2+. The kinetics of Ca2+ release from the vesicles is exponential. Using the grapho-analytical subtraction method, the kinetic parameters of this reaction were determined. Myometrium sarcolemma contains endogenous Ca2+-calmodulin-dependent protein kinase and phosphoprotein phosphatase which is inhibited by sodium o-vanadate. The Ca2+-calmodulin-dependent phosphorylation stimulates passive Ca2+ release from sarcolemmal vesicles. In the course of phosphorylation the capacity of the pool providing for rapid Ca2+ release increases by 61%, the initial rate of Ca2+ release showing a 28% increase. Trifluoroperazine, an inhibitor of Ca2+-calmodulin-dependent processes, eliminates the activating effect of phosphorylation on the rate of Ca2+ release from sarcolemmal vesicles.     

The relationship between extramitochondrial Ca2+ concentration, respiratory rate, and membrane potential in mitochondria from brown adipose tissue of the rat

The ability of isolated mitochondria from rat brown-adipose tissue to regulate extramitochondrial Ca2+ (measured by arsenazo) was studied in relation to their ability to produce heat (measured polarographically). The energetic state of the mitochondria was expressed as a membrane potential, delta psi (estimated with safranine), and was varied semi-physiologically by the use of different GDP concentrations. In these mitochondria GDP binds to the 32-kDa polypeptide, thermogenin, which regulates coupling. Ca2+ uptake (at 5 microM extramitochondrial Ca2+) was maximal at delta psi greater than 150 mV. Basal Ca2+ release increased from 1 to 2 nmol x min-1 x mg-1 below 150 mV. Na+ -stimulated rate of Ca2+ release was stable within the investigated delta psi span (100-160 mV). Initial Ca2+ levels were maintained below 0.2 microM for 100 mV less than delta psi less than 160 mV. Ca2+ levels maintained after Ca2+ challenge (20 nmol Ca2+ x mg-1) were below 0.4 microM for delta psi greater than 135 mM. Respiration was unstimulated for delta psi greater than 150 mV and was maximal at delta psi less than or equal to 135 mV. In the presence of well-oxidised substrates, the respiration at maximally activated thermogenin was markedly below fully uncoupled respiration and was probably limited by thermogenin activity--i.e. by a limited H+ reentry (OH- exit) and therefore by a membrane potential maintained at about 135 mV. It is concluded that at membrane potentials of 135 mV and above the mitochondria exhibit full Ca2+ control and are able to regulate thermogenic output up to maximum without interfering with this Ca2+ control. Membrane potential probably does not decrease below 135 mV in vivo. Therefore, Ca2+ homeostasis and thermogenesis are non-interfering and can be hormonally independently regulated, e.g. by alpha-adrenergic and beta-adrenergic stimuli, respectively.     

Effect of oxytocin on the calcium pump in myometrium sarcolemma

Oxytocin (10(-7) M) administered inside the myometrium sarcolemma vesicles closed outward by the cytoplasmic side is shown to inhibit Mg2+, ATP-dependent Ca2+ accumulation in these structures having no effect on the passive release of cation out of them. According to these results and to the data available in literature on the inhibitory action of the peptide hormone on Mg2+, Ca2+-ATPase of myometrium sarcolemma a conclusion is drawn that oxytocin inhibits the Ca pump activity in plasma membranes of the myometrium cells.     

The effect of oxytocin and sigetin on Ca2+ transport in the fraction of plasma membranes of myometrial cells

Oxytocin and sigetin were studied for their effect on the active and passive transport of Ca2+ in the fraction of myometrium sarcolemma in women. Oxytocin (5.10(-7) M) introduced into the sarcolemma vesicles and sigetin (5.10(-3) M) added into the incubation medium inhibit Mg2+, ATP-dependent accumulation of Ca2+ in these structures. The both agents in the mentioned concentration do not affect the passive release of cation from vesicles. A conclusion is drawn that inhibition of the calcium pump of myometrium cell plasma membranes underlies the physiological action of oxytocin and sigetin as stimulators of the contractile activity of the myometrium.     

Characterization of the plasma and mitochondrial membrane potentials of alveolar type II cells by the use of ionic probes

The lipophilic cation triphenylmethylphosphonium (TPMP+) and the potassium analog Rb+, were used to monitor the membrane potential (delta psi) of freshly isolated rabbit type II alveolar epithelial cells. Type II cells were found to accumulate TPMP+ rapidly at 37 degrees C in Hanks' balanced-salt solution with 5 microM tetraphenyl boron, but this accumulation was partially due to non-membrane potential dependent binding of TPMP+ to the cell. Lysophosphatidylcholine (lysoPC) was found to abolish delta psi and permitted correction for bound TPMP+ or Rb+. TPMP+ remaining in the cell following correction for binding represents the sum of mitochondrial and plasma membrane potential dependent accumulation. The accumulation of Rb+ by the type II cell was found to be independent of the mitochondrial membrane potential and indicated a trans-plasma membrane Rb+ distribution potential of -62.9 +/- 4 mV. A similar value was obtained by estimating the plasma membrane potential dependent accumulation of TPMP+ in type II cells whose mitochondria were depolarized with carbonylcyanide m-chlorophenylhydrazone (CCCP). The release of TPMP+ due to CCCP treatment also permitted an estimation for the trans-mitochondrial membrane potential of -141.8 +/- 10 mV. These techniques of membrane potential measurements were found to be sensitive to changes in delta psi induced by a number of inhibitors and ionophores. The ability to measure the membrane potential of the type II pneumocyte, and the changes caused by various agents, should be useful in characterizing the functional responses of this pulmonary surfactant producing cell.     

Electrochemical proton gradient in Micrococcus lysodeikticus cells and membrane vesicles.

Using the distribution of weak acids to measure the pH gradient (delta pH; interior alkaline) and the distribution of the lipophilic cation [3H]tetraphenylphosphonium+ to monitor the membrane potential (delta psi; interior negative), we studied the electrochemical gradient or protons (delta mu- H+) across the membrane of Micrococcus lysodeikticus cells and plasma membrane vesicles. With reduced phenazine methosulfate as electron donor, intact cells exhibited a relatively constant delta mu- H+ (interior negative and alkaline) of -193 mV to -223 mV from pH 5.5 to pH 8.5. On the other hand, in membrane vesicles under the same conditions, delta mu- H+ decreased from a maximum value of -166 mV at pH 5.5 to -107 mV at pH 8.0 and above. This difference is related to a differential effect of external pH on the components of delta mu- H+. In intact cells, delta pH decreased from about -86 mV (i.e., 1.4 units) at pH 5.5 to zero at pH 7.8 and above, and the decreases in delta pH was accompanied by a reciprocal increase in delta psi from -110 mV at pH 5.5 to -211 mV at pH 8.0 and above. In membrane vesicles, the decrease in delta pH with increasing external pH was similar to that described for intact cells; however, delta psi increased from -82 mV at pH 5.5 to only -107 mV at pH 8.0 and above.     

Coupling of Li+ distribution to the plasma membrane potential of rat cortical synaptosomes

The effect of the plasma membrane potential delta psi p on the transport rate and steady state distribution of Li+ was assessed in rat cortical synaptosomes. Up to 15 mM Li+ failed to saturate Li+ influx into polarized synaptosomes in a Na+-based medium with 3 mM external K+. Veratridine increased and tetrodotoxin, ouabain, or high external K+ decreased the rate of Li+ influx. At steady state, Li+ was concentrated about 3-fold in resting synaptosomes at 0.3 to 1 mM Li+ externally. Subsequent depolarization of the plasma membrane by veratridine or high external K+ induced an immediate release of Li+. When graded depolarizations were imposed onto the plasma membrane by varying concentrations of ouabain, veratridine, or external K+, steady state distribution of Li+ was linearly related with K+ distribution or electrochemical activity coefficients. It was concluded that uptake rate and steady state distribution of Li+ depend significantly on delta psi p. However, Li+ gradients were lower than predicted from delta psi p, suggesting that (secondary) active transport systems counteracted passive equilibration by uphill extrusion of Li+. The electrochemical potential difference delta mu Li+ maintained at a delta psi p of -72 mV was calculated to 4.2 kJ/mol of Li+. At physiological external K+, Li+ was not actively transported by the sodium pump. The ouabain sensitivity resulted from the coupling of Li+ uptake to the pump-dependent K+ diffusion potential. In low K+ and K+-free media, however, active transport of Li+ by the sodium pump contributed to total uptake. In the absence of K+, Li+ substituted for K+ in generating a delta psi p of -64 mV maximally, as calculated from TPMP+ distribution at 40 mM external Li+. Since Li+ gradients were far too low to account for a diffusion potential, it was assumed that Li+ gave rise to an electrogenic pump potential.     

Delta pH-induced transport of Ca 2+ into smooth muscle plasma membrane vesicle fraction

Using the fluorescent dye acridine orange, the feasibility of formation in myometrium sarcolemma of closed inside-out oriented vesicles and of a proton gradient created by the pH-jump method and stable in time, was demonstrated. At the initial value of delta pH = 2, the characteristic time of the gradient dissipation providing for the pH change by one unity is 4 to 5 minutes. The proton gradient oriented from the intravesicular space to the environment stimulated the Ca2+ influx into the vesicles. The transmembrane gradient of H+ with the inside-out oriented sarcolemmal vesicles prevents the Ca2+ influx. It is concluded that plasma membranes of smooth muscle cells contain alongside with the ATP- and Na(+)-dependent Ca2+ transport systems also a mechanism of the delta pH-induced transport of this bivalent cation.     

A study of passive Ca2+ flow through the sarcoplasmic reticulum of skeletal muscles. I. Passive Ca2+ efflux from vesicle membranes

The same level of passively loaded Ca2+ was observed both in the heavy (enriched in terminal cisternae) and light (enriched in longitudinal reticulum) sarcoplasmic reticulum (SR) fractions. The level of passively loaded Ca2+ of the both SR fractions decreased in the presence of 150 mM K+. However the rate and extent of Ca2+ release was greater from heavy SR fraction. The rate of Ca2+ release under conditions of antiport of K+, Na+, choline+ and gluconate-, Cl-, SCH- increased proportion with their permeability through the SR membrane. The initial rate of Ca2+ release also became higher under equal concentration of monovalent cation chloride both inside and outside the SR vesicles. Apparently, in this case Ca2+ release occurs through Ca-channels which are open at a membrane potential.     

Hydrogenosomes in the rumen protozoon Dasytricha ruminantium Schuberg.

The vacuo-lysosomes of Hevea brasiliensis (rubber tree) constitute a suitable model system for the study of active transport and energization at the level of the membrane of plant vacuoles. The pH gradient (delta pH) and the membrane potential (delta psi) of vacuo-lysosomes were determined by means of the weak base methylamine and the lipophilic cation tetraphenylphosphonium. The values obtained depended strongly on the experimental conditions such as medium pH or K+ concentration. Under experimental conditions, i.e., pH 7.5 outside and low K+, the delta pH amounts to about 0.9 unit, interior acid, and the delta psi to -120 mV, interior negative. The delta psi is presumably caused by the imposed K+ gradient, and the internal acidification might be a consequence of the passive proton inflow along the electric field. This explanation is sustained by the ineffectiveness of carbonyl cyanide p-trifluoromethoxyphenylhydrazone in destroying the delta pH and delta psi, whereas higher K+ concentration decreased both. Under conditions existing in vivo, the membrane potential might be significantly lower. The presence of ATP increased the acidification of the intravesicular space by 0.5pH unit to a delta pH of up to 1.4 and shifts the membrane potential at least 60mV to a more positive value. The change of the protonmotive potential did not occur with ADP; the pH-dependence of the change was identical with the pH-dependence of a vacuo-lysosomal membrane-bound ATPase, and the effect of ATPase was prevented by the presence of the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone. The change of protonmotive potential difference, brought about by the ATPase, was at least 90 mV. This is evidence that a vacuo-lysosomal ATPase in plants can function as an electrogenic proton pump that transfers protons into the vacuo-lysosomal space.     

Passive Ca2+ permeability of vesicular sarcolemmal preparations from myocardium

Vesicular preparations of sarcolemma isolated from rat myocardium possessed high ATPase (4.32 +/0 0.57 micromole/min per mg), adenylate cyclase (121 +/- 11 pmole/min per mg) and creatine kinase (1.74 +/- 0.35 micromole/min per mg) activities and a Na-Ca exchange activity specific for sodium. The ATPase activity was inhibited by digitoxigenin by 50-70% and was not changed by ouabain, EGTA, ionophore A23187 and oligomycin, thus showing the absence of mitochondrial and sarcoplasmic reticulum contaminations in the sarcolemmal preparations. The preparations consisted mostly of closed inside-out vesicles. The preparation was used to study the mechanism of Ca2+ penetration across the sarcolemmal membrane. For this purpose the vesicles were load with 45Ca2+, which relatively slowly diffused from the medium into the vesicles, and which was bound to the binding sites inside the vesicles (n = 20.5 +/- 4.6 nmoles per mg of protein, Kd approximately equal to 1.8 +/- 0.21 mM). The transmembrane movement of Ca2+ was demonstrated by the following findings: 1) the ionophore A23187 only insignificantly increased the total vesicular Ca2+ content, but strongly accelerated Ca2+ efflux from the vesicles along its concentration gradient; 2) gramicidin and osmotic shock caused a similar acceleration of Ca2+ efflux. Ca2+ efflux from these vesicles along Ca2+ concentration gradient was studied under conditions, when the extravesicular Ca2+ content was lowered due to its binding to EGTA and by dilution. The gradient of Ca2+ concentration was from 2.0 mM inside to approximately 0.1 micro M outside. The rate of 45Ca2+ efflux depended hyperbolically on the intravesicular Ca2+ efflux from the vesicles was inhibited by Mn2+, Co2+ and verapamil when they acted from the inside of the vesicles. An increase in ionophore A23187 concentration increased the efflux of Ca2+ hyperbolically and enhanced only the maximal rate of the efflux. It is concluded that the passive permeability of Ca2+ across the sarcolemmal membrane along its concentration gradient is controlled by Ca2+ binding to the membrane.     

Membrane potential of mitochondria in intact lymphocytes during early mitogenic stimulation.

The mitochondrial membrane potential (delta psi m) in intact lymphocytes was calculated by measuring the distribution of radiolabelled methyltriphenylphosphonium cation. The value obtained was 120 mV. The pH gradient across the mitochondrial membrane in situ (delta pH m) was estimated to be 73 mV (1.2 pH units). Thus the electrochemical gradient of protons was about 190 mV. Addition of the mitogen concanavalin A did not alter delta psi m, showing that, if movement of Ca2+ across the inner membrane of lymphocyte mitochondria occurs when concanavalin A is added, it is accompanied by charge-compensating ion movements.     

Potential-dependent passive transport of calcium in myocardium sarcolemma vesicles

The effect of membrane potential on passive Ca2+ transport in isolated cardiac sarcolemmal vesicles was investigated. The membrane potentials were induced by creating potassium gradients across the vesicular membranes in the presence of valinomycin. The fluorescence changes in the voltage-sensitive dye, dis-C3(5), were consistent with the induction of potassium equilibrium potentials. The rate of 45Ca2+ efflux from inside-out vesicles was considerably greater at 0 than at -80 or +55 mV; prepolarization of the membrane to +90 mV did not enhance the 45Ca2+ efflux upon subsequent depolarization. The voltage-dependent 45Ca2+ efflux increased with a rise in internal Ca2+ concentration and exhibited a saturation effect. Furthermore, evaluation of the rate of 45Ca2+ efflux over a wide range of membrane potentials produced a profile similar to that of current-voltage relationships for single calcium channels in isolated cardiomyocytes. It is concluded that the voltage-dependent Ca2+ efflux from the vesicles occurs via Ca2+-channels.     

Contribution of the Mg2+-, ATP- AND Na+-dependent Ca2+-transport systems to the regulation of Ca2+ concentration in myometrial cells

Studies with sarcolemma from cattle myometrium containing inside-out cytoplasmic vesicles, using Ca2+-EGTA buffer, showed that the affinity of ionized Ca2+ for the Mg2+- or ATP-dependent transport is higher than that for the Na+-Ca2+ exchange system (Kd = 3,2 X 10(-6) and (4.3-5.3) X 10(-5) M), respectively. The Km values for MgATP are 2.15 mM. Oxytocin added to the homogenization medium containing rabbit and cattle myometrium cells, i.e. during the formation of closed sarcolemmal fragments, resulted in inhibition of Mg2+, ATP-dependent accumulation of 45Ca2+ by plasma membranes. However, an addition of oxytocin to the incubation medium did not affect the kinetics of active accumulation of Ca2+. It was assumed that the system of non-electrogenic Na+-Ca2+ exchange in the myometrium possessing a low affinity for Ca2+ provides for the maintenance of ionized Ca2+ concentration in the myocytes at 10(-5) M. Therefore, this system cannot induce relaxation of mechanical tension of the uterus. Further decrease of Ca2+ in the myoplasm from 10(-5) to 10(-7) M and, correspondingly, the relaxation of myometrium is provided for by the Mg2+, ATP-dependent efflux of Ca2+ from the myocytes having a high affinity for this cation. The decrease of the activity of ATP-dependent Ca2+-pump by oxytocin is the cause of Ca2+ elevation in the myoplasm and, consequently, of myometrium contraction.     

The effects of membrane potential and lanthanides on the conformation of the Ca2+-transport ATPase in sarcoplasmic reticulum.

The effects of Ca2+, lanthanide ions (Gd3+, La3+ and Pr3+) and membrane potential on the fluorescence of tryptophan and covalently bound fluorescein were analysed in native and fluorescein isothiocyanate (FITC)-labelled sarcoplasmic reticulum vesicles. The binding of Ca2+ and lanthanides to the Ca2+-ATPase increases the fluorescence intensity of tryptophan and decreases the fluorescence intensity of FITC; the dependence of these effects on cation concentration is consistent with the involvement of the high-affinity Ca2+-binding sites of the Ca2+-ATPase in the cation-induced fluorescence changes. The fluorescence of FITC-labelled sarcoplasmic reticulum vesicles is also influenced by membrane potential changes induced by ion substitution. Inside positive potential increases, while inside negative potential decreases, the fluorescence of bound FITC. Smaller potential-dependent changes in tryptophan fluorescence were also observed. The effects of Ca2+, lanthanides and membrane potential on the fluorescence of tryptophan and FITC are discussed in terms of the two major conformations of the Ca2+-ATPase (E1 and E2), that are assumed to alternate during Ca2+ transport. The observations support the suggestion [Dux, Taylor, Ting-Beall & Martonosi (1985) J. Biol. Chem. 260, 11730-11743] that the vanadate-induced crystals of Ca2+-ATPase represent the E2, while the Ca2+ and lanthanide-induced crystals the E1, conformation of the enzyme.     

Mechanism of passive Ca2+ permeability of vesicular sarcolemmal preparations from rat hearts

Vesicular sarcolemmal preparations isolated from rat hearts were characterized by high total ATPase (4.32 +/- 0.57 mumol/min per mg), adenylate cyclase (121 +/- 11 pmol/min per mg) and creatine kinase (1.73 +/- 0.35 mumol/min per mg) activities as well as Na-Ca exchange specific to sodium. ATPase activity was inhibited with digitoxigenin by 50-70% and was not changed by ouabain, ionophore A23187 or oligomycin. Sarcolemmal vesicles bound [3H]digitoxigenin and [3H]ouabain in isotonic medium in the presence of Pi and Mg2+. The number of binding sites for hydrophobic digitoxigenin (N = 237 pmol/mg) was several-times higher than that for hydrophilic ouabain (N = 32.7 pmol/mg). These data show that sarcolemmal preparations were not significantly contaminated by mitochondria and sarcoplasmic reticulum and consisted mostly of inside-out vesicles. Incubation of these vesicles with 45Ca2+ (0.5-10 mM) led to penetration of the latter into the vesicles with the following binding characteristics: number of binding sites (N = 20.5 +/- 4.6 nmol/mg, Kd approximately equal to 2.0 mM). Ca2+ binding to the inner surface of vesicles was proved by the following facts: (1) Ca2+ ionophore A23187 increased slightly total intravesicular Ca2+ content but markedly accelerated Ca2+ efflux along its concentration gradient; (2) gramicidin and osmotic shock showed a similar accelerating effect. Ca2+ efflux from the vesicles along its concentration gradient ([Ca2+]i/[Ca2+]e = 2.0 mM/0.1 microM) was inhibited by Mn2+, Co2+, and verapamil when they acted inside the vesicles. The rate of Ca2+ efflux was hyperbolically dependent on intravesicular Ca2+ concentration (Km approximately equal to 2.9 mM). These data reveal that Ca2+ efflux from sarcolemmal vesicles is controlled by Ca2+ binding to the sarcolemmal membrane. Ca2+ efflux from the vesicles was stimulated 1.7--times after incubation of vesicles with 0.2 mM MgATP or MgADP and 15-times after treatment with 0.2 mM adenylyl beta, gamma-imidodiphosphate. Enhancement in the rate of Ca2+ efflux correlated with the increase in the intravesicular Ca2+ content. ATP-stimulated Ca2+ efflux was suppressed by verapamil and was nonmonotonically dependent upon the transmembrane potential created by the K+ concentration gradient in the presence of valinomycin, Ca2+ efflux being slower at extreme values of membrane potential (+/- 80 mV).