Ca2+ Binding to Site I of the Cardiac Ca2+ Pump Is Sufficient to Dissociate Phospholamban

Phospholamban (PLB) inhibits the activity of SERCA2a, the Ca²⁺-ATPase in cardiac sarcoplasmic reticulum, by decreasing the apparent affinity of the enzyme for Ca²⁺. Recent cross-linking studies have suggested that PLB binding and Ca²⁺ binding to SERCA2a are mutually exclusive. PLB binds to the E2 conformation of the Ca²⁺-ATPase, preventing formation of E1, the conformation that binds two Ca²⁺ (at sites I and II) with high affinity and is required for ATP hydrolysis. Here we determined whether Ca²⁺ binding to site I, site II, or both sites is sufficient to dissociate PLB from the Ca²⁺ pump. Seven SERCA2a mutants with amino acid substitutions at Ca²⁺-binding site I (E770Q, T798A, and E907Q), site II (E309Q and N795A), or both sites (D799N and E309Q/E770Q) were made, and the effects of Ca²⁺ on N30C-PLB cross-linking to Lys³²⁸ of SERCA2a were measured. In agreement with earlier reports with the skeletal muscle Ca²⁺-ATPase, none of the SERCA2a mutants (except E907Q) hydrolyzed ATP in the presence of Ca²⁺; however, all were phosphorylatable by P_i to form E2P. Ca²⁺ inhibition of E2P formation was observed only in SERCA2a mutants retaining site I. In cross-linking assays, strong cross-linking between N30C-PLB and each Ca²⁺-ATPase mutant was observed in the absence of Ca²⁺. Importantly, however, micromolar Ca²⁺ inhibited PLB cross-linking only to mutants retaining a functional Ca²⁺-binding site I. The dynamic equilibrium between Ca²⁺ pumps and N30C-PLB was retained by all mutants, demonstrating normal regulation of cross-linking by ATP, thapsigargin, and anti-PLB antibody. From these results we conclude that site I is the key Ca²⁺-binding site regulating the physical association between PLB and SERCA2a.     

Evidence for solvent-induced conformational changes of the soluble Dunaliella chloroplast coupling factor 1 (CF1)

The ATPase activity of the chloroplast coupling factor 1 (CF₁) isolated from the green alga Dunaliella is completely latent. A brief heat treatment irreversibly induces a Ca²⁺ -dependent activity. The Ca²⁺ dependent ATPase activity can be reversibly inhibited by ethanol, which changes the divalent cation dependency from Ca²⁺ to Mg²⁺. Both the Ca²⁺ -dependent and Mg²⁺ -dependent ATPase activities of heat-treated Dunaliella CF₁ are inhibited by monospecific antisera directed against Chlamydomonas reinhardi CF₁. However, when assayed under identical conditions, the Ca²⁺ -dependent ATPase activity is significantly more sensitive to inhibition by the antisera than is the Mg²⁺ -dependent activity. These data are interpreted as indicating that soluble Dunaliella CF₁ can exist in a variety of conformations, at least one of which catalyzes a Ca²⁺ -dependent ATPase and two or more of which catalyze an Mg²⁺ -dependent ATPase.     

Assembly of a Tyr122 Hydrophobic Cluster in Sarcoplasmic Reticulum Ca2+-ATPase Synchronizes Ca2+ Affinity Reduction and Release with Phosphoenzyme Isomerization

The mechanism whereby events in and around the catalytic site/head of Ca²⁺-ATPase effect Ca²⁺ release to the lumen from the transmembrane helices remains elusive. We developed a method to determine deoccluded bound Ca²⁺ by taking advantage of its rapid occlusion upon formation of E1PCa₂ and of stabilization afforded by a high concentration of Ca²⁺. The assay is applicable to minute amounts of Ca²⁺-ATPase expressed in COS-1 cells. It was validated by measuring the Ca²⁺ binding properties of unphosphorylated Ca²⁺-ATPase. The method was then applied to the isomerization of the phosphorylated intermediate associated with the Ca²⁺ release process E1PCa₂ → E2PCa₂ → E2P + 2Ca²⁺. In the wild type, Ca²⁺ release occurs concomitantly with EP isomerization fitting with rate-limiting isomerization (E1PCa₂ → E2PCa₂) followed by very rapid Ca²⁺ release. In contrast, with alanine mutants of Leu¹¹⁹ and Tyr¹²² on the cytoplasmic part of the second transmembrane helix (M2) and Ile¹⁷⁹ on the A domain, Ca²⁺ release in 10 μm Ca²⁺ lags EP isomerization, indicating the presence of a transient E2P state with bound Ca²⁺. The results suggest that these residues function in Ca²⁺ affinity reduction in E2P, likely via a structural rearrangement at the cytoplasmic part of M2 and a resulting association with the A and P domains, therefore leading to Ca²⁺ release.     

Aluminum-sensitive mutants of Saccharomyces cerevisiae

We are developing budding yeast, Saccharomyces cerevisiae, as a genetic system for the study of tolerance to the trivalent aluminum cation (Al³⁺). We have isolated eight mutants that are more sensitive to Al³⁺ than the wild type. Each mutant represented a different complementation group. A number of the mutants were pleiotropic, and showed defects in other stress responses, changes in tolerance to other metal cations, or abnormal morphology. Two mutants also showed increased dependence on supplemental Mg²⁺ and Ca²⁺. One mutant with a relatively specific sensitivity to Al³⁺ was chosen for molecular complementation. Normal Al³⁺ tolerance was restored by expression of the MAP kinase gene SLT2. Strains carrying deletions of the SLT2 gene, or of the gene for the corresponding MAP kinase–kinase SLK1, showed sensitivity to Al³⁺. These results indicate that the SLT2 MAP kinase signal transduction pathway is required for yeast to sense and respond to Al³⁺ stress.     

Regulation of the Cardiac Na+-Ca2+ Exchanger by the Endogenous XIP Region

The cardiac sarcolemmal Na⁺-Ca²⁺ exchanger is modulated by intrinsic regulatory mechanisms. A large intracellular loop of the exchanger participates in the regulatory responses. We have proposed (Li, Z., D.A. Nicoll, A. Collins, D.W. Hilgemann, A.G. Filoteo, J.T. Penniston, J.N. Weiss, J.M. Tomich, and K.D. Philipson. 1991. J. Biol. Chem. 266:1014–1020) that a segment of the large intracellular loop, the endogenous XIP region, has an autoregulatory role in exchanger function. We now test this hypothesis by mutational analysis of the XIP region. Nine XIP-region mutants were expressed in Xenopus oocytes and all displayed altered regulatory properties. The major alteration was in a regulatory mechanism known as Na⁺-dependent inactivation. This inactivation is manifested as a partial decay in outward Na⁺-Ca²⁺ exchange current after application of Na⁺ to the intracellular surface of a giant excised patch. Two mutant phenotypes were observed. In group 1 mutants, inactivation was markedly accelerated; in group 2 mutants, inactivation was completely eliminated. All mutants had normal Na⁺ affinities. Regulation of the exchanger by nontransported, intracellular Ca²⁺ was also modified by the XIP-region mutations. Binding of Ca²⁺ to the intracellular loop activates exchange activity and also decreases Na⁺-dependent inactivation. XIP-region mutants were all still regulated by Ca²⁺. However, the apparent affinity of the group 1 mutants for regulatory Ca²⁺ was decreased. The responses of all mutant exchangers to Ca²⁺ application or removal were markedly accelerated. Na⁺-dependent inactivation and regulation by Ca²⁺ are interrelated and are not completely independent processes. We conclude that the endogenous XIP region is primarily involved in movement of the exchanger into and out of the Na⁺-induced inactivated state, but that the XIP region is also involved in regulation by Ca²⁺.     

Sarcolemmal glucose transport in Ca2+-tolerant myocytes from adult rat heart. Calcium dependence of insulin action

Cardiac myocytes were isolated from adult rat ventricles by a method which preserves their functional integrity, including long survival in physiological concentrations of Ca²⁺. Sarcolemmal glucose transport was assessed by measuring linear initial uptake rates of the nonmetabolized glucose analog3-O-methyl-d-glucose. Transport was saturable and showed competition byd-glucose and other features of chemical and stereo-selectivity. Transport was stimulated by insulin in a dose-dependent manner, resulting in an almost 5-fold increase inV_max, with little change inK_m. Stimulation of 3-methylglucose transport by insulin was largely Ca²⁺ -dependent. Omission of Ca²⁺ from the incubation medium caused a minor rise in basal 3-methylglucose uptake but the insulin-stimulated rise inV_max was only 30%. The Ca²⁺ antagonist D600 also antagonized stimulation of hexose transport by insulin. In all the above respects, 3-methylglucose transport in myocytes is identical to that in intact heart muscle. In addition, the decrease in insulin response by Ca²⁺ emission was partially reversed by subsequent return to a Ca²⁺ -containing medium. ATP levels remained stable in the absence of Ca²⁺, showing that the Ca²⁺ dependence did not reflect nonspecific cell damage.     

Control of alpha-amylase mRNA accumulation by gibberellic Acid and calcium in barley aleurone layers

Pulse-labeling of barley (Hordeum vulgare L. cv Himalaya) aleurone layers incubated for 13 hours in 2.5 micromolar gibberellic acid (GA₃) with or without 5 millimolar CaCl₂ shows that α-amylase isozymes 3 and 4 are not synthesized in vivo in the absence of Ca²⁺. A cDNA clone for α-amylase was isolated and used to measure α-amylase mRNA levels in aleurone layers incubated in the presence and absence of Ca²⁺. No difference was observed in α-amylase mRNA levels between layers incubated for 12 hours in 2.5 micromolar GA₃ with 5 millimolar CaCl₂ and layers incubated in GA₃ alone. RNA isolated from layers incubated for 12 hours in GA₃ with and without Ca²⁺ was translated in vitro and was found to produce the same complement of translation products regardless of the presence of Ca²⁺ in the incubation medium. Immunoprecipitation of translation products showed that the RNA for α-amylase synthesized in Ca²⁺-deprived aleurone layers was translatable. Ca²⁺ is required for the synthesis of α-amylase isozymes 3 and 4 at a step after mRNA accumulation and processing.     

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.     

Essential Role of the EF-hand Domain in Targeting Sperm Phospholipase Cζ to Membrane Phosphatidylinositol 4,5-Bisphosphate (PIP2)

Sperm-specific phospholipase C-ζ (PLCζ) is widely considered to be the physiological stimulus that triggers intracellular Ca²⁺ oscillations and egg activation during mammalian fertilization. Although PLCζ is structurally similar to PLCδ1, it lacks a pleckstrin homology domain, and it remains unclear how PLCζ targets its phosphatidylinositol 4,5-bisphosphate (PIP₂) membrane substrate. Recently, the PLCδ1 EF-hand domain was shown to bind to anionic phospholipids through a number of cationic residues, suggesting a potential mechanism for how PLCs might interact with their target membranes. Those critical cationic EF-hand residues in PLCδ1 are notably conserved in PLCζ. We investigated the potential role of these conserved cationic residues in PLCζ by generating a series of mutants that sequentially neutralized three positively charged residues (Lys-49, Lys-53, and Arg-57) within the mouse PLCζ EF-hand domain. Microinjection of the PLCζ EF-hand mutants into mouse eggs enabled their Ca²⁺ oscillation inducing activities to be compared with wild-type PLCζ. Furthermore, the mutant proteins were purified, and the in vitro PIP₂ hydrolysis and binding properties were monitored. Our analysis suggests that PLCζ binds significantly to PIP₂, but not to phosphatidic acid or phosphatidylserine, and that sequential reduction of the net positive charge within the first EF-hand domain of PLCζ significantly alters in vivo Ca²⁺ oscillation inducing activity and in vitro interaction with PIP₂ without affecting its Ca²⁺ sensitivity. Our findings are consistent with theoretical predictions provided by a mathematical model that links oocyte Ca²⁺ frequency and the binding ability of different PLCζ mutants to PIP₂. Moreover, a PLCζ mutant with mutations in the cationic residues within the first EF-hand domain and the XY linker region dramatically reduces the binding of PLCζ to PIP₂, leading to complete abolishment of its Ca²⁺ oscillation inducing activity.     

H-mediated coupling of transmembrane Ca fluxes in vegetative Trichoderma viride mycelia suggested by the study of ageing and adaptation to extreme Ca concentrations

The adaptation to extreme concentrations of Ca²⁺ and its consequence on the properties of the ⁴⁵Ca²⁺ transport were studied in submerged mycelia of Trichoderma viride. The adaptation to low [Ca²⁺]_o did not cause changes in kinetic parameters of the ⁴⁵Ca²⁺ influx but the adaptation to high [Ca²⁺]_o increased the K_M(Ca2+). The V_max of the ⁴⁵Ca²⁺ influx decreased with the age of (non-adapted) mycelia with concomitant decrease of the K_M(Ca2+) these changes were prevented in mycelia adapted to high Ca²⁺. High [Ca²⁺]_o decreased the stimulation by the uncoupler, 3, 3′, 4′, 5-tetrachloro salicylanilide (TCS) (30 μM), as compared to the control, whereas the Ca²⁺ chelator, EGTA, stimulated it. In the aged mycelia, the stimulation by TCS of the ⁴⁵Ca²⁺ influx faded away, in parallel with the activity of the H⁺-ATPase. The ⁴⁵Ca²⁺ efflux from mycelia was affected by TCS in a similar way as the ⁴⁵Ca²⁺ influx. The results demonstrate the adaptive responses of transport processes participating in the mycelial Ca²⁺ homeostasis and ageing are in agreement with a notion that both Ca²⁺-influx and-efflux are coupled by the H⁺-homeostasis at the plasma membrane.     

Calcium-dependent Conformational Changes in Inositol Trisphosphate Receptors

We have used limited trypsin digestion and reactivity with PEG-maleimides (MPEG) to study Ca²⁺-induced conformational changes of IP₃Rs in their native membrane environment. We found that Ca²⁺ decreased the formation of the 95-kDa C-terminal tryptic fragment when detected by an Ab directed at a C-terminal epitope (CT-1) but not with an Ab recognizing a protected intraluminal epitope. This suggests that Ca²⁺ induces a conformational change in the IP₃R that allows trypsin to cleave the C-terminal epitope. Half-maximal effects of Ca²⁺ were observed at ∼0.5 μm and was sensitive to inhibition by IP₃. Ca²⁺ also stimulated the reaction of MPEG-5 with an endogenous thiol in the 95-kDa fragment. This effect was eliminated when six closely spaced cysteine residues proximal to the transmembrane domains were mutated (C2000S, C2008S, C2010S, C2043S, C2047S, and C2053S) or when the N-terminal suppressor domain (amino acids 1–225) was deleted. A cysteine substitution mutant introduced at the C-terminal residue (A2749C) was freely accessible to MPEG-5 or MPEG-20 in the absence of Ca²⁺. However, cysteine substitution mutants in the interior of the tail were poorly reactive with MPEG-5, although reactivity was enhanced by Ca²⁺. We conclude the following: a) that large conformational changes induced by Ca²⁺ can be detected in IP₃Rs in situ; b) these changes may be driven by Ca²⁺ binding to the N-terminal suppressor domain and expose a group of closely spaced endogenous thiols in the channel domain; and c) that the C-terminal cytosol-exposed tail of the IP₃R may be relatively inaccessible to regulatory proteins unless Ca²⁺ is present.     

Rat and Drosophila Synaptotagmin 4 Have Opposite Effects during SNARE-catalyzed Membrane Fusion

Synaptotagmins (Syt) are a large family of proteins that regulate membrane traffic in neurons and other cell types. One isoform that has received considerable attention is SYT4, with apparently contradictory reports concerning the function of this isoform in fruit flies and mice. SYT4 was reported to function as a negative regulator of neurotrophin secretion in mouse neurons and as a positive regulator of secretion of a yet to be identified growth factor from muscle cells in flies. Here, we have directly compared the biochemical and functional properties of rat and fly SYT4. We report that rat SYT4 inhibited SNARE-catalyzed membrane fusion in both the absence and presence of Ca²⁺. In marked contrast, fly SYT4 stimulated SNARE-mediated membrane fusion in response to Ca²⁺. Analysis of chimeric molecules, isolated C2 domains, and point mutants revealed that the C2B domain of the fly protein senses Ca²⁺ and is sufficient to stimulate fusion. Rat SYT4 was able to stimulate fusion in response to Ca²⁺ when the conserved Asp-to-Ser Ca²⁺ ligand substitution in its C2A domain was reversed. In summary, rat SYT4 serves as an inhibitory isoform, whereas fly SYT4 is a bona fide Ca²⁺ sensor capable of coupling Ca²⁺ to membrane fusion.     

Maturation in liver mitochondria of Ruthenium Red-sensitive calcium-ion-transport activity and the influence of glucagon administration in vivo and in utero

The maturation of Ca²⁺ transport in mitochondria isolated from rat liver was examined, from 5 days before birth. The mitochondria used were isolated from liver homogenates by centrifugation at 22000g-min. Ca²⁺ transport by mitochondria isolated from foetal liver is energy-dependent and Ruthenium Red-sensitive. The transmembrane pH gradient in these mitochondria is higher by about 7mV and the membrane potential lower by about 20mV than in adult mitochondria. The inclusion of 2mm-P_i in the incubation medium enhances the protonmotive force by approx. 30mV. The rate of Ca²⁺ influx in foetal mitochondria measured in buffered KCl plus succinate is low until about 2–3h after birth, when it increases to about 60% of adult values; approx. 24h later it has reached near-adult values. Higher rates of Ca²⁺ influx are observed in the presence of 2mm-P_i; 3–5 days before birth the rates are about one-third of adult values and decline slightly as birth approaches. By 2–3h post partum they have reached adult values. The inclusion of 12.5μm-MgATP with the P_i stimulates further the initial rate of Ca²⁺ influx in foetal mitochondria. The rates observed are constant over the prenatal period examined and are 50–60% of those observed in adult mitochondria. Mitochondria isolated from foetal livers 4–5 days before birth retain the accumulated Ca²⁺ for about 50min in the presence of 2mm-P_i. In the period 2 days before birth to birth, this ability is largely lost, but by 2–3h after birth Ca²⁺ retention is similar to that of adult mitochondria. The presence of 12.5μm-MgATP progressively enhances the Ca²⁺ retention time as development proceeds until 2–3h after birth, when it becomes less sensitive to added MgATP. Glucagon administration to older foetuses in utero enhances both the rate of mitochondrial Ca²⁺ influx assayed in the presence of 2mm-P_i and the time for which mitochondria retain accumulated Ca²⁺ in the presence of 12.5μm-MgATP and 2mm-P_i. Its administration to neonatal animals leads to an increase in mitochondrial Ca²⁺ retention similar to that seen in adult mitochondria. The data provide evidence that the Ruthenium Red-sensitive Ca²⁺ transporter is potentially as active in foetal mitochondria 5 days before birth as it is in adult mitochondria. They also show that foetal mitochondria have an ability to retain accumulated Ca²⁺ reminiscent of mitochondria from tumour cells and from hormone-challenged rat liver.     

Calcium Transport in Protoplasts Isolated from ml-o Barley Isolines Resistant and Susceptible to Powdery Mildew

Free cytoplasmic calcium has been postulated to play a role in preventing powdery mildew in a series of homozygous ml-o mutants of barley, Hordeum vulgare L. Protoplasts isolated from 7-day-old plants of the ml-o resistant-susceptible (R-S) barley isolines, Riso 5678/3^* × Carlsberg II R and S, were used to test for differences in fluxes of Ca²⁺ across the plasmalemma. Greater influx or lesser efflux might account for a higher free cytosolic Ca²⁺ postulated to exist in ml-o R mutants. Uniform patterns of uptake were maintained for 3 hours from solutions of 0.2 and 2 millimolar Ca²⁺. Washout curves of ⁴⁵Ca²⁺ from R and S protoplasts revealed three compartments—presumed to represent release from the vacuole, organelles, and the cytoplasm (which included bound as well as free Ca²⁺). Uptake and washout did not differ between isolines. On the basis of recent determinations of submicromolar levels of free cytoplasmic Ca²⁺ and our initial rates of ⁴⁵Ca-labeled Ca²⁺ uptake, we show that measurement of the unidirectional influx of Ca²⁺ across the plasmalemma is not feasible because the specific activity of the pool of free cytoplasmic calcium increases almost instantaneously to a level that would result in a significant, but unknown, efflux of label. Similarly, measurement of the efflux of Ca²⁺ across the plasmalemma is not possible since the activity of the pool of free cytoplasmic calcium is a factor of 350 smaller than the most rapid component of the washout experiment. This pool of cytoplasmic free Ca²⁺ will wash out too rapidly and be too small to detect under the conditions of these experiments.     

Calcium transport by ehrlich ascites cell mitochondria in vitro and in situ

The rate, maximum extent of accumulation, and passive release of Ca²⁺ by mitochondria within Ehrlich ascites tumor cells treated with digitonin and by isolated tumor mitochondria have been compared. The mitochondrial protein content of Ehrlich cells was determined by cytochrome and cytochrome oxidase analyses. The Ca²⁺ uptake rate in situ is approximately one-half the rate in vitro whereas maximum Ca²⁺ accumulation by mitochondria within the cell is about twice the value for isolated mitochondria. When isolated tumor mitochondria were supplemented with exogenous ATP the maximum uptake (approximately 3.0 μeq Ca²⁺/mg protein) was about the same as in situ. Adenine nucleotides retained in digitonized cells may account for the observed differences. The rate of uncoupler stimulated Ca²⁺ release from mitochondria within the cell (ca. 10 neq Ca²⁺/min · mg mitochondrial protein for Ca²⁺ loads up to 800 neq Ca²⁺/mg protein) agrees exceptionally well with previous estimates for isolated tumor mitochondria. Therefore the capacity for extensive Ca²⁺ accumulation without uncoupling and attenuation of Ca²⁺ efflux are virtually the same in the cell as in vitro.     

Calcium signalling in Drosophila photoreceptors measured with GCaMP6f

Drosophila phototransduction is mediated by phospholipase C leading to activation of cation channels (TRP and TRPL) in the 30000 microvilli forming the light-absorbing rhabdomere. The channels mediate massive Ca²⁺ influx in response to light, but whether Ca²⁺ is released from internal stores remains controversial. We generated flies expressing GCaMP6f in their photoreceptors and measured Ca²⁺ signals from dissociated cells, as well as in vivo by imaging rhabdomeres in intact flies. In response to brief flashes, GCaMP6f signals had latencies of 10–25 ms, reached 50% F_max with ∼1200 effectively absorbed photons and saturated (ΔF/F₀ ∼ 10–20) with 10000–30000 photons. In Ca²⁺ free bath, smaller (ΔF/F₀ ∼4), long latency (∼200 ms) light-induced Ca²⁺ rises were still detectable. These were unaffected in InsP₃ receptor mutants, but virtually eliminated when Na⁺ was also omitted from the bath, or in trpl;trp mutants lacking light-sensitive channels. Ca²⁺ free rises were also eliminated in Na⁺/Ca²⁺ exchanger mutants, but greatly accelerated in flies over-expressing the exchanger. These results show that Ca²⁺ free rises are strictly dependent on Na⁺ influx and activity of the exchanger, suggesting they reflect re-equilibration of Na⁺/Ca²⁺ exchange across plasma or intracellular membranes following massive Na⁺ influx. Any tiny Ca²⁺ free rise remaining without exchanger activity was equivalent to <10 nM (ΔF/F₀ ∼0.1), and unlikely to play any role in phototransduction.     

Quantitative aspects of drug-receptor interactions. I. Ca2+ and cholinergic receptor activation in smooth muscle: a basic model for drug-receptor interactions

An attempt has been made to devise a general model of drug-receptor interactions as it relates to the initiation of mechanical responses. A key feature of this model is the regulatory role played by membrane-bound Ca²⁺ (Ca_mem²⁺).The effects on the mechanical responsiveness of guinea pig ileal longitudinal muscle of four muscarinic agonists derived from and including the highly active cis-2-methyl-4-dimethylaminomethyl-1, 3-dioxolane methiodide have been studied. The concentration-response (isotonic contraction) curves of these four agonists at normal Ca_ext²⁺-levels show evidence of cooperativity (n_H > 1) and this was found to increase dramatically with decreasing [Ca_ext²⁺]. A three step model has been proposed, based on that previously advanced by Hurwitz &; Suria (1971), in which activation of the acetylcholine receptor initiates a Ca²⁺ translocation mechanism supplying the contractile machinery with Ca²⁺. Arguments are advanced to suggest that two sources of Ca²⁺ are thus utilized: membrane-bound (Ca_mem²⁺) and free extracellular (Ca_ext²⁺), the former being responsible for the initial phasic contraction and the latter for the slower phase of contractile development.Analysis of the theoretical model shows that the cooperativity of the concentration-response relationships derives not from the initial agonist-receptor interaction but from the subsequently initiated Ca²⁺ translocation step so that [Ca_int²⁺] ∝ [Ca_ext²⁺]ⁿ. The limiting value of n is found to be 6 and to be the same for agonists and partial agonists. According to this model intrinsic activity is determined by the linkage between the agonist-receptor complex and the Ca²⁺ translocation process.The general findings of this work are discussed in terms of an equilibrium between Ca²⁺-associated and Ca²⁺-dissociated membrane states. The similarities to other Ca²⁺ dependent processes are emphasized.     

Purification and properties of ATPase from an alkalophilic Bacillus

ATPase was purified from an alkalophilic Bacillus. The enzyme has a molecular weight of 410,000 and consists of five types of subunits of molecular weights of 60,000 (α), 58,000 (β), 34,000 (γ), 14,000 (δ), and 11,000 (?). The subunit structure is suggested to be α₃β₃γδ?. The enzyme is activated by Mg²⁺ and Ca²⁺. The pH optima of the enzyme with 0.1 and 2.0 mm Mg²⁺ are 9 and 6, and those with 1 and 10 mm Ca²⁺ are 8–9 and 7, respectively. Ca²⁺-ATPase hydrolyzes only ATP, whereas Mg²⁺-ATPase hydrolyzes GTP and, to a lesser extent, ATP. The values of V and K_m of the enzyme with ATP in the presence of 10 mm Ca²⁺ or 0.6 mm Mg²⁺ at pH 7.2 are 17 or 0.5 units/mg protein and 1.2 or 0.3 mm, respectively. The enzyme with Mg²⁺ is appreciably activated by HCO^?₃. Relationship of the ATPase to the active transport system in the bacterium is suggested.     

The role of inositol 1,4,5-trisphosphate in mobilizing calcium from intracellular stores in the salivary glands of Amblyomma americanum (L.)

Isolated tick salivary glands, permeabilized with digitonin in the presence of the Ca²⁺ uptake inhibitors, sodium azide and vanadate, released Ca²⁺ in response to 20 μM inositol-1,4,5-trisphosphate (IP₃). Inositol-1-phosphate (IP₁) and inositol-1,4-bisphosphate (IP₂) appeared to stimulate an uptake of Ca²⁺ into whole glands. Inositol-1,4,5-trisphosphate caused release of Ca²⁺ from a 100,000 g microsome enriched pellet; however, IP₁ and IP₂ were ineffective in stimulating an uptake or efflux of Ca²⁺. The combined 900 and 11,500 g pellets showed no significant release of Ca²⁺ in response to addition of IP₃. Inositol-1,4,5-trisphosphate concentrations as low as 1 μM are capable of stimulating a significant release of Ca²⁺ from microsomes. Results suggest that intracellular Ca²⁺ is mobilized from microsomal intracellular stores in response to agonists which increase cytosolic IP₃ in tick salivary glands. Results also suggest a possible role for IP₁ and IP₂ or both in stimulating an uptake of Ca²⁺ into vanadate and azide-insensitive intracellular pools.