Regulation of the rat sarcoplasmic reticulum calcium release channel by calcium

The regulation by calcium of the ryanodine receptor/SR calcium release channel (RyR) from rat skeletal muscle was studied under isolated conditions and in situ. RyRs were either solubilized and incorporated into lipid bilayers or single fibres were mounted into a Vaseline gap voltage clamp. Single channel data were compared to parameters determined from the calculated calcium release flux. With K⁺ (250 mM) being the charge carrier the single channel conductance was 529 pS at 50 M Ca²⁺ cis and trans, and decreased with increasing cis [Ca²⁺]. Open probability showed a bell shaped calcium dependence revealing an activatory and an inhibitory Ca²⁺ binding site (Hill coefficients of 1.18 and 1.28, respectively) with half activatory and inhibitory concentrations of 9.4 and 298 M. The parameters of the inhibitory site agreed with the calcium dependence of channel inactivation deduced from the decline in SR calcium release in isolated fibres. Mean open time showed slight [Ca²⁺] dependence following a single exponential at every Ca²⁺ concentration tested. Closed time histograms, at high [Ca²⁺], were fitted with three exponentials, from which the longest was calcium independent, and resembled the recovery time constant of SR inactivation (115 ± 15 ms) obtained in isolated fibres. The data are in agreement with a model where calcium binding to the inhibitory site on RyR would be responsible for the calcium dependent inactivation in situ.     

Presynaptic calcium channels and the depletion of synaptic cleft calcium ions

The entry of calcium ions (Ca(2+)) through voltage-gated calcium channels is an essential step in the release of neurotransmitter at the presynaptic nerve terminal. Because the calcium channels are clustered at the release sites, the flux of Ca(2+) into the terminal inevitably removes the ion from the adjacent extracellular space, the synaptic cleft. We have used the large calyx-type synapse of the chick ciliary ganglion to test for synaptic cleft Ca(2+) depletion. The terminal was voltage clamped at a holding potential (V(H)) of -80 mV and a depolarizing pulse was applied to a range of potentials (-60 to +60 mV). The voltage pulse activated a sustained inward calcium current and was followed, on return of the membrane potential to V(H), by an inward calcium tail current. The amplitude of the tail current reflects both the number of open calcium channels at the end of the voltage pulse and the Ca(2+) electrochemical gradient. External barium was substituted for calcium as the charge-carrying ion because initial experiments demonstrated calcium-dependent inactivation of the presynaptic calcium channels. Tail current recruitment was compared in calyx nerve terminals that remained attached to the postsynaptic neuron and therefore retained a synaptic cleft, with terminals that had been fully isolated. In isolated terminals, the tail currents exhibited recruitment curves that could be fit by a Boltzmann distribution with a mean V(1/2) of 0.4 mV and a slope factor of 5.4. However, in attached calyces tail current recruitment was skewed to depolarized potentials with a mean V(1/2) of 11.9 mV and a slope factor of 12.0. The degree of skew of the recruitment curve in the attached calyces correlated with the amplitude of the inward current evoked by the step depolarization. The simplest interpretation of these findings is that during the depolarizing pulse Ba(2+) is removed from the synaptic cleft faster than it is replenished, thus reducing the tail current by reducing the driving force for ion entry. Ca(2+) depletion during presynaptic calcium channel activation is likely to be a general property of chemical transmission at fast synapses that sets a functional limit to the duration of sustained secretion. The synapse may have evolved to minimized cleft depletion by developing a calcium-efficient mechanism to gate transmitter release that requires the concurrent opening of only a few low conductance calcium channels.     

Structurally delineating stromal interaction molecules as the endoplasmic reticulum calcium sensors and regulators of calcium release-activated calcium entry

Summary:  The endoplasmic reticulum (ER) lumen stores a crucial source of calcium (Ca²⁺) maintained orders of magnitude higher than the cytosol for the activation of a plethora of cellular responses transmitted in health and disease by a mutually efficient and communicative exchange of Ca²⁺ between compartments. A coordination of the Ca²⁺ signal is evident in the development of Ca²⁺ release-activated Ca²⁺ (CRAC) entry, vital to lymphocyte activation and replenishing of the ER Ca²⁺ stores, where modest decreases in ER luminal Ca²⁺ induce sustained increases in cytosolic Ca²⁺ sourced from steadfast extracellular Ca²⁺ supplies. While protein sensors that transduce Ca²⁺ signals in the cytosol such as calmodulin are succinctly understood, comparative data on the ER luminal Ca²⁺ sensors is only recently coming to light with the discovery that stromal interaction molecules (STIMs) sense variations in ER stored Ca²⁺ levels in the functional regulation of plasma membrane Orai proteins, the major component of CRAC channel pores. Drawing from data on the role of STIMs in the modulation of CRAC entry, this review illustrates the structural features that delimit the functional characteristics of ER Ca²⁺ sensors relative to well known cytoplasmic Ca²⁺ sensors.     

Changes in subcellular calcium transport in rat hearts during the calcium paradox

Changes in the transport of calcium in the sarcoplasmic reticulum (SR) and mitochondria (MT), of the rat heart during calcium paradox were investigated. Calcium binding and uptake by SR in the paradox hearts were from about 1.5 to 2 times greater than in normal hearts, whereas in the MT of the paradox hearts they were about half those of normal hearts. There was no difference between paradox and normal hearts in calcium stimulated ATPase activity in the SR. The ultrastructure of the MT was disrupted in calcium paradox, but the SR was essentially the same as in normal hearts. We propose that during calcium paradox, the intracellular calcium overload damages both calcium transport and the ultrastructure of the MT, but that calcium transport by the SR is accelerated to compensate for the calcium overload.     

Effects of calcium depletion and calcium paradox on the ultrastructure of the frog heart

The alterations in the ultrastructure of the isolated perfused Rana ridibunda hearts that were subjected to prolonged calcium depletion and reperfusion with calcium containing medium are described using thin section electron microscopy. Deprivation of calcium resulted in broadened intercellular spaces and in mild cell swelling. Cell to cell contact was maintained throughout calcium depletion, while myofibrils and mitochondria remained intact. Reintroduction of calcium containing buffers to calcium depleted hearts resulted in an irreversible injury of the frog myocardial cells. The main characteristics of the reperfusion induced damage were contraction band formation, distortion and degradation of the myofibrils, extensive swelling of the mitochondria and formation of intramitochondrial electron dense deposits. Mitochondrial aggregation, intermitochondrial junctions, expulsion of the mitochondria to the sarcolemmal membrane and peripheral condensation of nuclear chromatin were also observed. Our results indicate that frog myocardial cells show a marked resistance even to a prolonged calcium depletion, retaining their integrity and their contact. However, the following reperfusion greatly alters the ultrastructure of frog myocardium and the observed alterations are typical of the irreversible damage induced in calcium overload situations.     

Cytoplasmic calcium oscillations and store-operated calcium influx

Intracellular calcium oscillations have fascinated scientists for decades. They provide an important cellular signal which, unlike most signalling mechanisms, is digitally encoded. While it is generally agreed that oscillations most frequently arise from cyclical release and re-uptake of intracellularly stored calcium, it is becoming increasingly clear that influx of calcium across the plasma membrane also plays a critical role in their maintenance and even in delivering their signal to the correct cellular locus. In this review we will discuss the role played by Ca²⁺ entry mechanisms in Ca²⁺ oscillations, and approaches to understanding the molecular nature of this Ca²⁺ entry pathway.     

Extracellular calcium sensing and extracellular calcium signaling

The cloning of a G protein-coupled extracellular Ca(2+) (Ca(o)(2+))-sensing receptor (CaR) has elucidated the molecular basis for many of the previously recognized effects of Ca(o)(2+) on tissues that maintain systemic Ca(o)(2+) homeostasis, especially parathyroid chief cells and several cells in the kidney. The availability of the cloned CaR enabled the development of DNA and antibody probes for identifying the CaR's mRNA and protein, respectively, within these and other tissues. It also permitted the identification of human diseases resulting from inactivating or activating mutations of the CaR gene and the subsequent generation of mice with targeted disruption of the CaR gene. The characteristic alterations in parathyroid and renal function in these patients and in the mice with "knockout" of the CaR gene have provided valuable information on the CaR's physiological roles in these tissues participating in mineral ion homeostasis. Nevertheless, relatively little is known about how the CaR regulates other tissues involved in systemic Ca(o)(2+) homeostasis, particularly bone and intestine. Moreover, there is evidence that additional Ca(o)(2+) sensors may exist in bone cells that mediate some or even all of the known effects of Ca(o)(2+) on these cells. Even more remains to be learned about the CaR's function in the rapidly growing list of cells that express it but are uninvolved in systemic Ca(o)(2+) metabolism. Available data suggest that the receptor serves numerous roles outside of systemic mineral ion homeostasis, ranging from the regulation of hormonal secretion and the activities of various ion channels to the longer term control of gene expression, programmed cell death (apoptosis), and cellular proliferation. In some cases, the CaR on these "nonhomeostatic" cells responds to local changes in Ca(o)(2+) taking place within compartments of the extracellular fluid (ECF) that communicate with the outside environment (e.g., the gastrointestinal tract). In others, localized changes in Ca(o)(2+) within the ECF can originate from several mechanisms, including fluxes of calcium ions into or out of cellular or extracellular stores or across epithelium that absorb or secrete Ca(2+). In any event, the CaR and other receptors/sensors for Ca(o)(2+) and probably for other extracellular ions represent versatile regulators of numerous cellular functions and may serve as important therapeutic targets.     

Changes in subcellular calcium transport in rat hearts during the calcium paradox.

Changes in the transport of calcium in the sarcoplasmic reticulum (SR) and mitochondria (MT), of the rat heart during calcium paradox were investigated. Calcium binding and uptake by SR in the paradox hearts were from about 1.5 to 2 times greater than in normal hearts, whereas in the MT of the paradox hearts they were about half those of normal hearts. There was no difference between paradox and normal hearts in calcium stimulated ATPase activity in the SR. The ultrastructure of the MT was disrupted in calcium paradox, but the SR was essentially the same as in normal hearts. We propose that during calcium paradox, the intracellular calcium overload damages both calcium transport and the ultrastructure of the MT, but that calcium transport by the SR is accelerated to compensate for the calcium overload.     

Regulation of calcium release by calcium inside the sarcoplasmic reticulum in ventricular myocytes

 To study the effects of changes in sarcoplasmic reticulum (SR) intraluminal Ca²⁺ on the Ca²⁺ release mechanism, we correlated the activity of single cardiac ryanodine receptor (RyR) channels, monitored in planar bilayers, with the properties of spontaneous elementary Ca²⁺ release events (sparks) in intact ventricular myocytes, monitored by scanning confocal microfluorimetry. Under both normal conditions and Ca²⁺ overload, induced by elevation of extracellular [Ca²⁺], Ca²⁺ sparks represented single populations of events. During Ca²⁺ overload, the frequency of sparks increased from 0.8 to 3.1 events per second per 100 μm line scanned, and their amplitude increased from 100 nM to 400 nM. The duration of the Ca²⁺ sparks, however, was not altered. Changes in the properties of Ca²⁺ sparks were accompanied by only an ≈ 30% increase in the SR Ca²⁺ content, as determined by emptying the intracellular Ca²⁺ stores using caffeine. When single Ca²⁺ release channels were incorporated into lipid bilayers and activated by cytoplasmic Ca²⁺ (≈ 100 nM) and ATP (3 mM), elevation of Ca²⁺ on the luminal side from 20 μM to 0.2–20 mM resulted in a 1.2-fold to 7-fold increase, respectively, in open probability (P _o). This potentiation of P _o was due to an increase in mean open time and frequency of events. The relative effect of luminal Ca²⁺ was greater at low levels of cytoplasmic [Ca²⁺] than at high levels of cytoplasmic [Ca²⁺], and no effect of luminal Ca²⁺ was observed to occur in channels activated by 0.5–50 μM cytoplasmic Ca²⁺ in the absence of ATP. Our results suggest that SR Ca²⁺ release channels are modulated by SR intraluminal Ca²⁺. These alterations in properties of release channels may account for, or contribute to, the mechanism of spontaneous Ca²⁺ release in cardiac myocytes Received: 15 May 1996 / Received after revision: 5 June 1996 / Accepted: 8 July 1996     

Mechanotransduction pathways in bone: calcium fluxes and the role of voltage-operated calcium channels

Changes in strain distribution across the vertebrate skeleton induce modelling and remodelling of bone structure. This relationship, like many in biomedical science, has been recognised since the 1800s, but it is only the recent development of in vivo and in vitro models that is allowing detailed investigation of the cellular mechanisms involved. A number of secondary messenger pathways have been implicated in load transduction by bone cells, and many of these pathways are similar to those proposed for other load-responsive cell types. It appears that load transduction involves interaction between several messenger pathways, rather than one specific switch. Interaction between these pathways may result in a cascade of responses that promote and maintain bone cell activity in remodelling of bone. The paper outlines research on the early rapid signals for load transduction and, in particular, activation of membrane channels in osteoblasts. The involvement of calcium channels in the immediate load response and the modulation of intracellular calcium as an early signal are discussed. These membrane channels present a possible target for manipulation in the engineering of bone tissue repair.     

Mechanotransduction pathways in bone: calcium fluxes and the role of voltage-operated calcium channels

Changes in strain distribution across the vertebrate skeleton induce modelling and remodelling of bone structure. This relationship, like many in biomedical science, has been recognised since the 1800s, but it is only the recent development of in vivo and in vitro models that is allowing detailed investigation of the cellular mechanisms involved. A number of secondary messenger pathways have been implicated in load transduction by bone cells, and many of these pathways are similar to those proposed for other load-responsive cell types. It appears that load transduction involves interaction between several messenger pathways, rather than one specific switch. Interaction between these pathways may result in a cascade of responses that promote and maintain bone cell activity in remodelling of bone. The paper outlines research on the early rapid signals for load transduction and, in particular, activation of membrane channels in osteoblasts. The involvement of calcium channels in the immediate load response and the modulation of intra-cellular calcium as an early signal are discussed. These membrane channels present a possible target for manipulation in the engineering of bone tissue repair.     

Dependence on calcium concentration and stoichiometry of the calcium pump in human red cells

1. In resealed human red cells loaded with Ca-EGTA buffer solutions it was found that the intracellular free Ca(2+) concentration for half saturation of the Ca transport system (which pumps Ca out of the cell) is equal to or smaller than 4 x 10(-6)M and thus closely agrees with the dissociation constant of the Ca + Mg activated membrane ATPase.2. The maximal rate of Ca transport from resealed cells to medium was found to be 0.148 +/- 0.009 mumole/ml. cells.min at 28 degrees C.3. The rate of Ca transport was unaffected by a variation of the extracellular Ca(2+) concentration from 3.10(-7) to 5.10(-3)M.4. Evidence is presented making it probable that the stoichiometric relation between Ca transported and ATP hydrolysed is 1:1 rather than 2:1.5. As the Ca transport is quite rapid even at half saturation and the passive leak for Ca negligible in intact cells it can be predicted that the steady-state cellular Ca(2+) concentration must be low, most probably less than 10(-6) mumole/ml. cells. Transport from cells containing 5.10(-7) mumole/ml. into blood plasma is thermodynamically compatible with the normal plasma Ca(2+) concentration and the normal cellular ATP, ADP and P(i) content.6. Treatment with the mercurial PCMBS in the cold for 15 hr allows to load red cells with 1 mumole Ca/ml. cells without destroying their ability to transport Ca after removal of the mercurial.7. It is shown that at high cellular Ca concentrations (0.1-3 mumole/ml. cells) about 50% of the total is free Ca(2+) on account of binding mainly to dialysable cell constituents.     

Effect of previous dietary history of calcium intake on the skeleton and calcium absorption in the rat

1. Rats were fed either a high or a low calcium diet for 6 weeks. Thereafter all were given the low calcium diet, and the time during which differences persisted between the two groups in apparent absorption of calcium and urinary excretion of calcium has been studied. The apparent absorption of calcium is the same in the two groups after 15 days but the differences in urinary excretion of calcium persist longer.2. Faecal endogenous calcium and true absorption of calcium have been measured by an isotope dilution technique. The fall in apparent absorption of calcium seen in rats fed a high calcium diet is in part due to a fall in true absorption. Faecal endogenous calcium is also increased in these animals.3. The amount of calcium and phosphate in the bones is increased by feeding a high calcium diet and decreased by feeding a very low phosphate diet. Femur, humerus, caudal vertebrae and calvaria are equally affected by the different diets.4. There are strong inverse correlations between absorption of calcium and the percentage of calcium in the bones. This suggests that absorption of calcium may be regulated by the degree of bone mineralization.5. Rats were fed diets with different calcium phosphate ratios for 6 weeks. Thereafter all were given a low calcium diet with a calcium: phosphorous ratio of 1 and absorption and urinary excretion of calcium were studied. The effects of the different diets may be explained by their effect on calcium status and bone mineralization.