Control of membrane fusion by phospholipid head groups II. The role of phosphatidylethanolamine in mixtures with phosphatidate and phosphatidylinositol

Membrane fusion induced by Ca²⁺ and Mg²⁺ in large unilamellar vesicles composed of mixtures of phosphatidylethanolamine with phosphatidate and phosphatidylinositol was studied by means of a fluorescence assay for the intermixing of internal aqueous contents of the vesicles. The threshold concentrations of Ca²⁺ or Mg²⁺ required for fusion increased only moderately when up to 80 mol% phosphatidylethanolamine was included with phosphatidate at pH 7.4, but no fusion could be detected in vesicles containing 70 mol% phosphatidylcholine even at high concentrations of Ca²⁺ or Mg²⁺. Phosphatidate-phosphatidylethanolamine (1 : 4) vesicles could be induced to fuse by 0.1 mM Ca²⁺ in the presence of a Mg²⁺ concentration which alone was insufficient for fusion. When equimolar amounts of phosphatidylethanolamine was included with phosphatidylinositol, the vesicles were susceptible to fusion by Ca²⁺, although pure phosphatidylinositol vesicles themselves merely aggregate and do not fuse (Sundler, R. and Papahadjopoulos, D. (1981) Biochim. Biophys. Acta 649, 743–750, accompanying paper). The role of phosphatidylethanolamine acyl chains, and hence the possible involvement of the bilayer-hexagonal (H_II) transition in membrane fusion, was examined by the temperature dependence of Ca²⁺-induced fusion in phosphatidylinositol-dimyristoylphosphatidylethanolamine (1 : 1) vesicles. Fusion was strictly dependent on the gel-liquid crystalline transition of the mixture and not on the phase behavior of the phosphatidylethanolamines. Comparable fusion rates were obtained for both egg yolk phosphatidylethanolamine and dimyristoylphosphatidylethanolamine at 50°C. As the dimyristoylphosphatidylethanolamine does not convert to a non-bilayer phase in this temperature range, we conclude that the bilayer-hexagonal transition is not necessary for membrane fusion. We propose that the dehydration characteristics of the phospholipids and their metal ion complexes are the critical factors determining fusion suceptibility of phospholipid membranes.     

Divalent cation influx and calcium homeostasis in germinal vesicle mouse oocytes

Prior to maturation, mouse oocytes are arrested at the germinal vesicle (GV) stage during which they experience constitutive calcium (Ca²⁺) influx and spontaneous Ca²⁺ oscillations. The oscillations cease during maturation but Ca²⁺ influx continues, as the oocytes’ internal stores attain maximal content at the culmination of maturation, the metaphase II stage. The identity of the channel(s) that underlie this Ca²⁺ influx has not been completely determined. GV and matured oocytes are known to express three Ca²⁺ channels, Ca_V3.2, TRPV3 and TRPM7, but females null for each of these channels are fertile and their oocytes display minor modifications in Ca²⁺ homeostasis, suggesting a complex regulation of Ca²⁺ influx. To define the contribution of these channels at the GV stage, we used different divalent cations, pharmacological inhibitors and genetic models. We found that the three channels are active at this stage. Ca_V3.2 and TRPM7 channels contributed the majority of Ca²⁺ influx, as inhibitors and oocytes from homologous knockout (KO) lines showed severely reduced Ca²⁺ entry. Sr²⁺ influx was promoted by Ca_V3.2 channels, as Sr²⁺ oscillations were negligible in Ca_V3.2-KO oocytes but robust in control and Trpv3-KO GV oocytes. Mn²⁺ entry relied on expression of Ca_V3.2 and TRPM7 channels, but Ni²⁺ entry depended on the latter. Ca_V3.2 and TRPV3 channels combined to fill the Ca²⁺ stores, although Ca_V3.2 was the most impactful. Studies with pharmacological inhibitors effectively blocked the influx of divalent cations, but displayed off-target effects, and occasionally agonist-like properties. In conclusion, GV oocytes express channels mediating Ca²⁺ and other divalent cation influx that are pivotal for fertilization and early development. These channels may serve as targets for intervention to improve the success of assisted reproductive technologies.     

Polymorphic phase behaviour of cardiolipin from bovine heart and from Bacillus subtilis as detected by 31P-NMR and freeze-fracture techniques: Effects of Ca2+, Mg2+, Ba2+ and temperature

The structures formed by aqueous dispersions of cardiolipin isolated from bovine heart and B. subtilis have been studied by ³¹P-NMR and freeze-fracture electron microscopy. The sodium salts of both cardiolipins form bilayers. The Ca²⁺, Mg²⁺ and Ba²⁺ salts undergo well-defined bilayer → hexagonal (H_II) transitions, the temperature of which is dependent on the cation involved and the fatty acid composition of the cardiolipin.     

Selectivity signatures of three isoforms of recombinant T-type Ca channels

Voltage-gated Ca²⁺ channels (VGCCs) are recognized for their superb ability for the preferred passage of Ca²⁺ over any other more abundant cation present in the physiological saline. Most of our knowledge about the mechanisms of selective Ca²⁺ permeation through VGCCs was derived from the studies on native and recombinant L-type representatives. However, the specifics of the selectivity and permeation of known recombinant T-type Ca²⁺-channel α1 subunits, Ca_v3.1, Ca_v3.2 and Ca_v3.3, are still poorly defined. In the present study we provide comparative analysis of the selectivity and permeation Ca_v3.1, Ca_v3.2, and Ca_v3.3 functionally expressed in Xenopus oocytes. Our data show that all Ca_v3 channels select Ca²⁺ over Na⁺ by affinity. Ca_v3.1 and Ca_v3.2 discriminate Ca²⁺, Sr²⁺ and Ba²⁺ based on the ion's effects on the open channel probability, whilst Ca_v3.3 discriminates based on the ion's intrapore binding affinity. All Ca_v3s were characterized by much smaller difference in the K_D values for Na⁺ current blockade by Ca²⁺ (K_D1 ∼ 6 μM) and for Ca²⁺ current saturation (K_D2 ∼ 2 mM) as compared to L-type channels. This enabled them to carry notable mixed Na⁺/Ca²⁺ current at close to physiological Ca²⁺ concentrations, which was the strongest for Ca_v3.3, smaller for Ca_v3.2 and the smallest for Ca_v3.1. In addition to intrapore Ca²⁺ binding site(s) Ca_v3.2, but not Ca_v3.1 and Ca_v3.3, is likely to possess an extracellular Ca²⁺ binding site that controls channel permeation. Our results provide novel functional tests for identifying subunits responsible for T-type Ca²⁺ current in native cells.     

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.     

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.     

Simulation of intracellular Ca2+ oscillation in a sympathetic neurone

Three different theoretical models were considered for the mechanism of the oscillation of the intracellular free Ca²⁺ ([Ca²⁺]_i) linked to the K⁺ conductance of the plasma membrane (G_K) observed in bullfrog sympathetic ganglion cells. The models assumed a Ca²⁺-induced Ca²⁺ release mechanism, an active Ca²⁺ uptake mechanism at a Ca²⁺ reservoir site in the ganglion cell, and a Michaelis—Menten type relationship between [Ca²⁺]_i and G_K. Including both active and passive Ca²⁺ transport mechanisms at the plasma membrane, either a one-compartment model or a two-compartment model for the intracellular Ca²⁺ store reconstructed successfully the [Ca²⁺]_i oscillation and rhythmic membrane hyperpolarizations observed in the ganglion cell, and simulated most of their characteristics. On the other hand, a two-compartment model disregarding of Ca²⁺ transport at the plasma membrane failed to reproduce the oscillations of [Ca²⁺]_i and membrane potential.     

Ca binding to sarcoplasmic reticulum ATPase phosphorylated by Pi reveals four thapsigargin-sensitive Ca sites in the presence of ADP

Sarcoplasmic reticulum (SR) Ca²⁺-ATPase was phosphorylated by P_i at pH 8.0 in the presence of dimethyl sulfoxide (Me₂SO). Under these conditions, it was possible to measure transient ⁴⁵Ca²⁺ binding to the phosphoenzyme. Binding reached 1.2 Ca²⁺ per phosphoenzyme (E-PCa_x) within 10 min in 30% Me₂SO, 20 mM MgCl₂ and 0.1 mM P_i and the phosphoenzyme only decreased by 23% during this period. This Ca²⁺ binding was abolished by thapsigargin, showing that it is associated with functional sites of the Ca²⁺-ATPase. At 40% Me₂SO, simultaneous addition of Ca²⁺ and ADP increased Ca²⁺ binding up to almost four Ca²⁺ per phosphoenzyme (ADPE-PCa_y), revealing a species bearing simultaneously four Ca²⁺ sites. Both E-PCa_x and ADPE-PCa_y were further identified as distinct species by (2′,3′-O-2(2,4,6-trinitrophenyl)adenosine 5′-triphosphate) fluorescence, which revealed long-range modifications in the Ca²⁺-transport sites induced by ADP binding to E-P. In addition, E-PCa_x was shown to be a functional intermediate of the cycle leading to ATP synthesis provided that Me₂SO was diluted. These findings indicate that more than two functional Ca²⁺-sites exist on the functional Ca²⁺-ATPase unit, and that the additional sites become accessible upon ADP addition. This is compatible with a four-site model of the SR Ca²⁺-ATPase allowing simultaneous binding of Ca²⁺ at lumenal and cytosolic sites. The stoichiometries for Ca²⁺ binding found here could either be interpreted as binding of four Ca²⁺ on a Ca²⁺-ATPase monomer considered as the functional unit or as binding of two Ca²⁺ per monomer of a functional dimer.     

Ca2+-mobility in the sarcoplasmic reticulum of ventricular myocytes is low

The sarcoplasmic reticulum (SR) in ventricular myocytes contains releasable Ca²⁺ for activating cellular contraction. Recent measurements of intra-SR (luminal) Ca²⁺ suggest a high diffusive Ca²⁺-mobility constant (D_CaSR). This could help spatially to unify SR Ca²⁺-content ([Ca²⁺]_SRT) and standardize Ca²⁺-release throughout the cell. But measurements of localized depletions of luminal Ca²⁺ (Ca²⁺-blinks), associated with local Ca²⁺-release (Ca²⁺-sparks), suggest D_CaSR may actually be low. Here we describe a novel method for measuring D_CaSR. Using a cytoplasmic Ca²⁺-fluorophore, we estimate regional [Ca²⁺]_SRT from localized, caffeine-induced SR Ca²⁺-release. Caffeine microperfusion of one end of a guinea pig or rat myocyte diffusively empties the whole SR at a rate indicating D_CaSR is 8-9 μm²/s, up to tenfold lower than previous estimates. Ignoring background SR Ca²⁺-leakage in our measurement protocol produces an artifactually high D_CaSR (>40 μm²/s), which may also explain the previous high values. Diffusion-reaction modeling suggests that a low D_CaSR would be sufficient to support local SR Ca²⁺-signaling within sarcomeres during excitation-contraction coupling. Low D_CaSR also implies that [Ca²⁺]_SRT may readily become spatially nonuniform, particularly under pathological conditions of spatially nonuniform Ca²⁺-release. Local control of luminal Ca²⁺, imposed by low D_CaSR, may complement the well-established local control of SR Ca²⁺-release by Ca²⁺-channel/ryanodine receptor couplons.     

Effects of cations on the plasma membrane of Blastocladiella emersonii zoospores. An ESR study

The physical properties of the plasma membrane of the aquatic phycomycete Blastocladiella emersonii were investigated, in particular the effects of cations on membrane structure. Intact zoospores and lipid extracts were labelled with the spin-labels 5-nitroxystearate (5-NS), 12-nitroxystearate (12-NS), and 2,2,6,6-tetramethylpiperidine-1-oxyl (Tempo). Electron spin resonance spectroscopy indicated a total of three breaks in plots of the hyperfine splitting parameter, 2T_|, order parameter, S, and the partition coefficient, f, vs. temperature. The first and third break points (T_L and T_H) were found to be independent of the external K⁺, Ca²⁺, or Mg²⁺ concentrations. They were similar to the break points found in aqueous dispersions of lipid extracts and correlate well with the temperature limits for zoospore viability. In contrast, the middle break point (T_M) was markedly influenced by the external Ca²⁺ concentration. Ca²⁺ increased T_M from 12°C (no Ca²⁺ added) to 22°C (10 mM Ca²⁺), i.e., growth temperature. K⁺ reversed this Ca²⁺ effect, downshifting T_M from 22°C to 10°C. A comparison of the physico-chemical effects of these ions on the membrane, as revealed by the cation-induced shift in T_M, is closely correlated with the temperature dependence and physiological effects of cations on zoospore differentiation. This suggests that cations may modify the physical state of the plasma membrane and be involved in regulating the initial changes during zoospore encystment.     

Identification and Characterization of the Ca-ATPase which Drives Active Transport of Ca at the Plasma Membrane of Radish Seedlings

In microsomes from 24-hour-old radish (Raphanus sativus L.) seedlings ATP-dependent Ca²⁺ uptake occurs only in inside-out plasma membrane vesicles (F Rasi-Caldogno, MC Pugliarello, MI De Michelis [1987] Plant Physiol 83: 994-1000). A Ca²⁺-dependent ATPase activity can be shown in the same microsomes, when assays are performed at pH 7.5. The Ca²⁺-dependent ATPase is stimulated by the Ca²⁺ ionophore A₂₃₁₈₇ and is localized at the plasma membrane. Ca²⁺-dependent ATPase activity and ATP-dependent Ca²⁺ uptake present very similar saturation kinetics with erythrosin B (50% inhibition at about 0.1 micromolar), free Ca²⁺ (half-maximal rate at about 70 nanomolar), and MgATP (K_m 15-20 micromolar). Ca²⁺ uptake can be sustained by GTP or ITP at about 60% the rate measured in the presence of ATP; only very low Ca²⁺ uptake is sustained by CTP or UTP and none by ADP. These results indicate that the Ca²⁺-ATPase described in this paper is the enzyme which drives active transport of Ca²⁺ at the plasma membrane of higher plants.     

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.     

The Ca2+-Regulation of the Mitochondrial External NADPH Dehydrogenase in Plants Is Controlled by Cytosolic pH

NADPH is a key reductant carrier that maintains internal redox and antioxidant status, and that links biosynthetic, catabolic and signalling pathways. Plants have a mitochondrial external NADPH oxidation pathway, which depends on Ca²⁺ and pH in vitro, but concentrations of Ca²⁺ needed are not known. We have determined the K_0.5(Ca²⁺) of the external NADPH dehydrogenase from Solanum tuberosum mitochondria and membranes of E. coli expressing Arabidopsis thaliana NDB1 over the physiological pH range using O₂ and decylubiquinone as electron acceptors. The K_0.5(Ca²⁺) of NADPH oxidation was generally higher than for NADH oxidation, and unlike the latter, it depended on pH. At pH 7.5, K_0.5(Ca²⁺) for NADPH oxidation was high (≈100 μM), yet 20-fold lower K_0.5(Ca²⁺) values were determined at pH 6.8. Lower K_0.5(Ca²⁺) values were observed with decylubiquinone than with O₂ as terminal electron acceptor. NADPH oxidation responded to changes in Ca²⁺ concentrations more rapidly than NADH oxidation did. Thus, cytosolic acidification is an important activator of external NADPH oxidation, by decreasing the Ca²⁺-requirements for NDB1. The results are discussed in relation to the present knowledge on how whole cell NADPH redox homeostasis is affected in plants modified for the NDB1 gene.     

Studies on the mechanism of regulation of the red-cell Ca2+ pump by calmodulin and ATP

(1) The effects of calmodulin binding on the rates of Ca²⁺-dependent phosphorylation and dephosphorylation of the red-cell Ca²⁺ pump, have been tested in membranes stripped of endogenous calmodulin or recombined with purified calmodulin. (2) In Mg²⁺-containing media, phosphorylation and dephosphorylation rates are accelerated by a large factor (at 0°C), but the steady-state level of phosphoenzyme is unaffected by calmodulin binding (at 0°C and 37°C). In Mg²⁺-free media, slower rates of phosphoenzyme formation and hydrolysis are observed, but both rates and the steady-state phosphoenzyme level are raised following calmodulin binding. (3) At 37°C and 0°C, the rate of (Ca²⁺ + Mg²⁺)-ATPase activity is stimulated maximally by 6–7-fold, following calmodulin binding. At 37°C the apparent Ca²⁺ affinity for sustaining ATP hydrolysis is raised at least 20-fold, K_m(Ca) ? 10 μM (—calmodulin) and K_m(Ca) < 0.5 μM (+ calmodulin), but at 0°C the apparent Ca²⁺ affinity is very high in calmodulin-stripped membranes and little or no effect of calmodulin is observed (K_m(Ca) ? 3–4 · 10^-8 M). (Ca²⁺ + Mg²⁺)-ATPase activity in calmodulin activated membranes and at saturating ATP levels, is sharply inhibited by addition of calcium in the range 50–2000 μM. (4) A systematic study of the effects of the nucleotide species MgATP, CaATP and free ATP on (Ca²⁺ + Mg²⁺)-ATPase activity in calmodulin-activated membranes reveals: (a) In the 1–10 μmolar concentration range MgATP, CaATP and free ATP appear to sustain (Ca²⁺ + Mg²⁺)-ATPase activity equally effectively. (b) In the range 100–2000 μM, MgATP accelerates ATP hydrolysis (K_m(MgATP) ? 360 μM), and CaATP is an inhibitor (K_i(CaATP) ? 165 μM), probably competing with MgATP fo the regulatory site. (5) The results suggest that calmodulin binding alters the conformational state of the Ca²⁺- pump active site, producing a high (Ca²⁺ + Mg²⁺)-ATPase activity, high Ca²⁺ affinity and regulation of activity by MgATP.     

Kinetics and ion specificity of Na/Ca exchange mediated by the reconstituted beef heart mitochondrial Na/Ca antiporter

The Na⁺/Ca²⁺ antiporter was purified from beef heart mitochondria and reconstituted into liposomes containing fluorescent probes selective for Na⁺ or Ca²⁺. Na⁺/Ca²⁺ exchange was strongly inhibited at alkaline pH, a property that is relevant to rapid Ca²⁺ oscillations in mitochondria. The effect of pH was mediated entirely via an effect on the K_m for Ca²⁺. When present on the same side as Ca²⁺, K⁺ activated exchange by lowering the K_m for Ca²⁺ from 2  to 0.9 μM. The K_m for Na⁺ was 8 mM. In the absence of Ca²⁺, the exchanger catalyzed high rates of Na⁺/Li⁺ and Na⁺/K⁺ exchange. Diltiazem and tetraphenylphosphonium cation inhibited both Na⁺/Ca²⁺ and Na⁺/K⁺ exchange with IC₅₀ values of 10 and 0.6 μM, respectively. The V_max for Na⁺/Ca²⁺ exchange was increased about fourfold by bovine serum albumin, an effect that may reflect unmasking of an autoregulatory domain in the carrier protein.     

CRAC channels in secretory epithelial cell function and disease

The receptor-evoked Ca²⁺ signal in secretory epithelia mediate many cellular functions essential for cell survival and their most fundamental functions of secretory granules exocytosis and fluid and electrolyte secretion. Ca²⁺ influx is a key component of the receptor-evoked Ca²⁺ signal in secretory cell and is mediated by both TRPC and the STIM1-activated Orai1 channels that mediates the Ca²⁺ release-activated current (CRAC) I_crac. The core components of the receptor-evoked Ca²⁺ signal are assembled at the ER/PM junctions where exchange of materials between the plasma membrane and internal organelles take place, including transfer of lipids and Ca²⁺. The Ca²⁺ signal generated at the confined space of the ER/PM junctions is necessary for activation of the Ca²⁺-regulated proteins and ion channels that mediate exocytosis with high fidelity and tight control. In this review we discuss the general properties of Ca²⁺ signaling, PI(4,5)P₂ and other lipids at the ER/PM junctions with regard to secretory cells function and disease caused by uncontrolled Ca²⁺ influx.     

Modeling local and global intracellular calcium responses mediated by diffusely distributed inositol 1,4,5-trisphosphate receptors

Considerable insight into intracellular Ca²⁺ responses has been obtained through the development of whole cell models that are based on molecular mechanisms, e.g., single channel kinetics of the inositol 1,4,5-trisphosphate (IP₃) receptor Ca²⁺ channel. However, a limitation of most whole cell models to date is the assumption that IP₃ receptor Ca²⁺ channels (IP₃Rs) are globally coupled by a “continuously stirred” bulk cytosolic [Ca²⁺], when in fact open IP₃Rs experience elevated “domain” Ca²⁺ concentrations. Here we present a 2N+2-compartment whole cell model of local and global Ca²⁺ responses mediated by N=100,000 diffusely distributed IP₃Rs, each represented by a four-state Markov chain. Two of these compartments correspond to bulk cytosolic and luminal Ca²⁺ concentrations, and the remaining 2N compartments represent time-dependent cytosolic and luminal Ca²⁺ domains associated with each IP₃R. Using this Monte Carlo model as a starting point, we present an alternative formulation that solves a system of advection-reaction equations for the probability density of cytosolic and luminal domain [Ca²⁺] jointly distributed with IP₃R state. When these equations are coupled to ordinary differential equations for the bulk cytosolic and luminal [Ca²⁺], a realistic but minimal model of whole cell Ca²⁺ dynamics is produced that accounts for the influence of local Ca²⁺ signaling on channel gating and global Ca²⁺ responses. The probability density approach is benchmarked and validated by comparison to Monte Carlo simulations, and the two methods are shown to agree when the number of Ca²⁺ channels is large (i.e., physiologically realistic). Using the probability density approach, we show that the time scale of Ca²⁺ domain formation and collapse (both cytosolic and luminal) may influence global Ca²⁺ oscillations, and we derive two reduced models of global Ca²⁺ dynamics that account for the influence of local Ca²⁺ signaling on global Ca²⁺ dynamics when there is a separation of time scales between the stochastic gating of IP₃Rs and the dynamics of domain Ca²⁺.     

TRPC5 Is a Ca2+-activated Channel Functionally Coupled to Ca2+-selective Ion Channels

TRPC5 forms non-selective cation channels. Here we studied the role of internal Ca²⁺ in the activation of murine TRPC5 heterologously expressed in human embryonic kidney cells. Cell dialysis with various Ca²⁺ concentrations (Ca²⁺_i) revealed a dose-dependent activation of TRPC5 channels by internal Ca²⁺ with EC₅₀ of 635.1 and 358.2 nm at negative and positive membrane potentials, respectively. Stepwise increases of Ca²⁺_i induced by photolysis of caged Ca²⁺ showed that the Ca²⁺ activation of TRPC5 channels follows a rapid exponential time course with a time constant of 8.6 ± 0.2 ms at Ca²⁺_i below 10 μm, suggesting that the action of internal Ca²⁺ is a primary mechanism in the activation of TRPC5 channels. A second slow activation phase with a time to peak of 1.4 ± 0.1 s was also observed at Ca²⁺_i above 10 μm. In support of a Ca²⁺-activation mechanism, the thapsigargin-induced release of Ca²⁺ from internal stores activated TRPC5 channels transiently, and the subsequent Ca²⁺ entry produced a sustained TRPC5 activation, which in turn supported a long-lasting membrane depolarization. By co-expressing STIM1 plus ORAI1 or the α₁C and β₂ subunits of L-type Ca²⁺ channels, we found that Ca²⁺ entry through either calcium-release-activated-calcium or voltage-dependent Ca²⁺ channels is sufficient for TRPC5 channel activation. The Ca²⁺ entry activated TRPC5 channels under buffering of internal Ca²⁺ with EGTA but not with BAPTA. Our data support the hypothesis that TRPC5 forms Ca²⁺-activated cation channels that are functionally coupled to Ca²⁺-selective ion channels through local Ca²⁺ increases beneath the plasma membrane.