Influence of caffeine,Ca2+, and Mg2+ on ryanodine depression of the tension transient in skinned myocardial fibers of the rabbit

Ryanodine, a blocker for Ca²⁺-release channels of the sarcoplasmic reticulum (SR Ca²⁺-release channels), induces depression of myocardial contraction in isolated intact muscle, which is consistent with depression of the caffeine-induced tension transient in skinned muscle fibers. In isolated SR, ryanodine binds to a specific receptor with high affinity, and this binding is enhanced by caffeine and increasing Ca²⁺ and decreased by increasing Mg²⁺. The aim of this study was to test the hypothesis that depression of myocardial contraction is mediated by changes in ryanodine-receptor binding properties. Accordingly, factors (caffeine, Ca²⁺, and Mg²⁺) affecting ryanodine-receptor binding properties in the isolated SR membrane were studied in skinned myocardial fibers from adult rabbits. The depression of the caffeine-induced tension transient by ryanodine (ryanodine depression) influenced by these three factors was measured. In a dose-dependent manner, increasing caffeine or Ca²⁺ concentrations enhanced the ryanodine depression. The concentrations for 50% ryanodine depression (IC₅₀) approximated 7mM for caffeine, and pCa 5.25 for Ca²⁺. When 1 M ryanodine and 25 mM caffeine were combined, ryanodine depression was independent of Ca²⁺ at low Ca²⁺ concentrations (20%–30% at pCa>8 and 7.5) and was a direct function of Ca²⁺ at higher concentrations (pCa 7.5–6.0 with IC₅₀ approx. pCa 6.75). In contrast, increasing Mg²⁺ reduced the ryanodine depression with IC₅₀ approximately equal to pMg 3.3. In conclusion, the caffeineor Ca²⁺-enhanced, and Mg²⁺-reduced ryanodine depression observed in this study is consistent with known ryanodinereceptor binding properties.     

Effect of chloride on Ca2+ release from the sarcoplasmic reticulum of mechanically skinned skeletal muscle fibres

 The effect of intracellular Cl^– on Ca²⁺ release in mechanically skinned fibres of rat extensor digitorum longus (EDL) and toad iliofibularis muscles was examined under physiological conditions of myoplasmic [Mg²⁺] and [ATP] and sarcoplasmic reticulum (SR) Ca²⁺ loading. Both in rat and toad fibres, the presence of 20 mM Cl^–in the myoplasm increased Ca²⁺ leakage from the SR at pCa (i.e. –log₁₀ [Ca²⁺]) 6.7, but not at pCa 8. Ca²⁺ uptake was not significantly affected by the presence of Cl^–. This Ca²⁺-dependent effect of Cl^– on Ca²⁺ leakage was most likely due to a direct action on the ryanodine receptor/Ca²⁺ release channel, and could influence channel sensitivity and the resting [Ca²⁺] in muscle fibres in vivo. In contrast to this effect, acute addition of 20 mM Cl^– to the myoplasm caused a 40–50% reduction in Ca²⁺ release in response to a low caffeine concentration both in toad and rat fibres. One possible explanation for this latter effect is that the addition of Cl^– induces a potential across the SR (lumen negative) which might reduce Ca²⁺ release via several different mechanisms. Received: 20 October 1997 / Received after revision: 1 December 1997 / Accepted: 2 December 1997     

Ruthenium red reduces the Ca2+ sensitivity of Ca2+ uptake into cardiac sarcoplasmic reticulum

 Ruthenium red inhibits mitochondrial Ca²⁺ uptake and is widely used as an inhibitor of ryanodine-sensitive Ca²⁺ channels that function to release Ca²⁺ from the sarcoplasmic reticulum (SR) of muscle cells. It also has effects on other Ca²⁺ channels and ion transporters. To study the effects of ruthenium red on Ca²⁺ transport into the SR of cardiac muscle cells, fluorescence measurements of Ca²⁺ uptake into cardiac SR vesicles were made. Ruthenium red significantly decreased the Ca²⁺ sensitivity of SR uptake in a dose-dependent manner at concentrations ranging from 5 μM to 20 μM. There were no significant effects of ruthenium red on the maximum velocity or the Hill coefficient of SR Ca²⁺ uptake. Received: 14 January 1998 / Received after revision: 12 March 1998 / Accepted: 16 March 1998     

Ca2+/CaM-dependent inactivation of the skeletal muscle L-type Ca2+ channel (Cav1.1)

Ca²⁺-dependent modulation via calmodulin (CaM) has been documented for most high-voltage-activated Ca²⁺ channels, but whether the skeletal muscle L-type channel (Ca_v1.1) exhibits this property has been unknown. In this paper, whole-cell current and fluorescent resonance energy transfer (FRET) recordings were obtained from cultured mouse myotubes to test for potential involvement of CaM in function of Ca_v1.1. When prolonged depolarization (800 ms) was used to evoke Ca_v1.1 currents in normal myotubes, the fraction of current remaining at the end of the pulse displayed classic signs of Ca²⁺-dependent inactivation (CDI), including U-shaped voltage dependence, maximal inactivation (~30%) at potentials eliciting maximal inward current, and virtual elimination of inactivation when Ba²⁺ replaced external Ca²⁺ or when 10 mM BAPTA was included in the pipette solution. Furthermore, CDI was virtually eliminated (from 30 to 8%) in normal myotubes overexpressing mutant CaM (CaM₁₂₃₄) that does not bind Ca²⁺, whereas CDI was unaltered in myotubes overexpressing wild-type CaM (CaM_wt). In addition, a significant FRET signal (E = 4.06%) was detected between fluorescently tagged Ca_v1.1 and CaM_wt coexpressed in dysgenic myotubes, demonstrating for the first time that these two proteins associate in vivo. These findings show that CaM associates with and modulates Ca_v1.1. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.     

The contribution of the sarcoplasmic reticulum Ca2+-transport ATPase to caffeine-induced Ca2+ transients of murine skinned skeletal muscle fibres

The present study was carried out to investigate the contribution of the Ca²⁺-transport ATPase of the sarcoplasmic reticulum (SR) to caffeine-induced Ca²⁺ release in skinned skeletal muscle fibres. Chemically skinned fibres of balb-C-mouse EDL (extensor digitorum longus) were exposed for 1 min to a free Ca²⁺ concentration of 0.36 μM to load the SR with Ca²⁺. Release of Ca²⁺ from the SR was induced by 30 mM caffeine and recorded as an isometric force transient. For every preparation a pCa/force relationship was constructed, where pCa = −log₁₀ [Ca²⁺]. In a new experimental approach, we used the pCa/force relationship to transform each force transient directly into a Ca²⁺ transient. The calculated Ca²⁺ transients were fitted by a double exponential function: Y ₀ + A ₁⋅exp (−t/t ₁) + A ₂⋅exp(t/t ₂), with A ₁ < 0 < A ₂, t ₁ < t ₂ and Y ₀, A ₁, A ₂ in micromolar. Ca²⁺ transients in the presence of the SR Ca²⁺-ATPase inhibitor cyclopiazonic acid (CPA) were compared to those obtained in the absence of the drug. We found that inhibition of the SR Ca²⁺-ATPase during caffeine-induced Ca²⁺ release causes an increase in the peak Ca²⁺ concentration in comparison to the control transients. Increasing CPA concentrations prolonged the time-to-peak in a dose-dependent manner, following a Hill curve with a half-maximal value of 6.5 ± 3 μM CPA and a Hill slope of 1.1 ± 0.2, saturating at 100 μM. The effects of CPA could be simulated by an extended three-compartment model representing the SR, the myofilament space and the external bathing solution. In terms of this model, the SR Ca²⁺-ATPase influences the Ca²⁺ gradient across the SR membrane in particular during the early stages of the Ca²⁺ transient, whereas the subsequent relaxation is governed by diffusional loss of Ca²⁺ into the bathing solution. Received: 2 February 1996/Accepted: 1 April 1996     

Pharmacological clues to calmodulin-mediated activation of skeletal ryanodine receptor using [3H]-ryanodine binding

The hypothesis that calmodulin (CaM) may act as a positive modulator of junctional SR Ca²⁺-release channel/ryanodine receptor (RyR1) rests largerly on the demonstrated capacity of CaM to interact structurally and functionally with RyR1 at pCa > 8 (Tripathy et al., 1995). The goal of the present [³H]-ryanodine binding study was to produce, in isolated terminal cisternae (TC) and in purified junctional face membrane (JFM), CaM-mediated activation of RyR1 at less extreme pCa values, i.e. closer to resting myoplasmic pCa, and to analyze more accurately the corresponding changes in binding affinity for ryanodine of the receptor. We were able to monitor these changes at an optimum pCa of 6.5, following pre-activation of native RyR1 by mM concentrations of caffeine or M concentrations of antraquinone compound doxorubicin, and at various doses of these triggers. CaM increased the affinity of ryanodine binding to isolated TC in the presence of 1 mM AMP–PCP as an activator of RyR1; the K _d for ryanodine binding was reduced from 21.8 nM to 13.2 nM by 1 M CaM. Similar effects of CaM were seen when AMP–PCP was replaced by either caffeine or doxorubicin. In order to discount the involvement of SR extra-junctional proteins in this effect, the experiments were repeated on purified JFM. Again, CaM increased the affinity of ryanodine binding; the K _d was reduced from 11.1 nM to 7.0 nM by 1 M CaM (in the presence of doxorubicin). Pharmacological triggers of CaM-activatory action on native RyR1, like caffeine and doxorubicin, have long been characterized for their ability to activate RyR1 by increasing the Ca²⁺-sensitivity of the receptor. We speculate that the triggering effect of these agents on the CaM-mediated mechanism in vitro might mimick one of the early effects of the activation of RyR1 in skeletal muscle, during E–C coupling.     

Effects of tetracaine and procaine on skinned muscle fibres depend on free calcium

Summary The local anaesthetics, tetracaine and procaine have previously been found to block, induce or potentiate Ca²⁺ release from the sarcoplasmic reticulum (SR) of skeletal muscle depending on the preparation, experimental conditions and design. We now show that low concentrations of tetracaine and procaine block SR Ca²⁺ release whereas high concentrations induce release from the SR of amphibian and mammalian skinned fibres. Both actions depend on pCa, such that a shift in pCa can alter their effect from blocking to releasing Ca²⁺. In skinned fibres with Ca²⁺-loaded SR, tetracaine (1mm) produced a tonic contraction with a time to half-peak of 15–20 s and a magnitude reaching 80% of maximum force. Ca²⁺ release by tetracaine or procaine occured at pCa 6.5 and was not blocked by Ruthenium Red (RR) (25 m). This action of tetracaine was attributed to SR Ca²⁺ release rather than to a displacement of bound Ca²⁺ because fibres lacking a functional SR due to pre-treatment with quercetin (100 m), A 23187 (100 g ml^–1) or Triton X-100 (1%) did not contract after additions of tetracaine. Lower concentrations of tetracaine (0.5mm) and procaine (10mm) blocked contractions due to caffeine (at pCa 6.73), sulphydryl oxidizing agents, or Ca²⁺-induced Ca²⁺ release (CICR). The inhibition of CICR as a function of pCa was difficult to measure quantitatively since lowering pCa to elicit CICR twitches was sufficient to initiate tetracaine-induced tonic contractions.Experiments with isolated SR vesicles showed that 1mm tetracaine inhibited CICR, over a wide range of pCa but 3–5mm tetracaine induced rapid Ca²⁺ release. The opposite effects of tetracaine and procaine depend mostly on their concentration in SR vesicles and/or pCa in skinned fibres. Blockade of release seems to occur via the CICR pathway, and induction of release through an increase in SR membrane permeability.Abbreviations SR sarcoplasmic reticulum - HEPES N-2-hydroxy-ethylpiperazine-N¹-2-ethanesulphonic acid - EGTA ethylene glycol bis (-aminoethyl ether)-N,N,N¹,-N¹-tetraacetic acid - CICR Ca²⁺-induced Ca²⁺ release - MOPS morpholinopropane sulphonic acid - RR Ruthenium Red     

Inositol trisphosphate enhances Ca2+ oscillations but not Ca2+-induced Ca2+ release from cardiac sarcoplasmic reticulum

The role of inositol 1,4,5-trisphosphate [Ins(1,4,5)P ₃] in excitation-contraction coupling in cardiac muscle is still unclear, although many laboratories are beginning to assume a critical role for this putative second messenger. Earlier studies from this laboratory [Nosek et al. (1986) Am J Physiol 250:C807] found that Ins(1,4,5)P ₃ enhanced spontaneous Ca²⁺ release and the caffeine sensitivity of Ca²⁺ release from myocardial sarcoplasmic reticulum (SR) and proposed an increase in the Ca²⁺ sensitivity of the release as a possible mechanism. In order to clarify the phyisological relevance of these actions of Ins(1,4,5)P ₃ and specifically to test the effect of Ins(1,4,5)P ₃ on the Ca²⁺ sensitivity of Ca²⁺ release, we compared the effects of Ins(1,4,5)P ₃ on Ca²⁺ oscillations and on Ca²⁺-induced Ca²⁺ release (CICR) from the SR in saponin-skinned rat papillary muscle. We found that: (a) 30 M Ins(1,4,5)P ₃ enhanced the Ca²⁺ oscillations (measured by tension oscillations) from the rat cardiac SR, consistent with the previous report on guinea pig tissue; (b) both GTP and GTP[S] enhanced Ca²⁺ oscillations. The effect was not additive to that of Ins(1,4,5)P ₃ indicating that two different Ca²⁺-release pools do not exist in cardiac SR; (c) 30 M Ins(1,4,5)P ₃ had no effect on the Ca²⁺ sensitivity of CICR; (d) Ins(1,4,5)P ₃ (up to 30 M) had no effect on SR Ca²⁺ loading. The studies were performed in the presence of Cd²⁺ or 2,3-bisphosphoglycerate, agents that inhibit Ins(1,4,5)P ₃ hydrolysis. These results suggest that: (a) two different mechanisms underlie Ca²⁺ oscillations and CICR, Ins(1,4,5)P ₃ influencing Ca²⁺ oscillations but not CICR; (b) Ins(1,4,5)P ₃ does not increase the Ca²⁺ sensitivity of Ca²⁺ release from the SR; (c) cardiac muscle is different from smooth muscle where Ca²⁺ release from the SR is dependent upon GTP; (d) the physiological role of Ins(1,4,5)P ₃ in excitation-contraction coupling in cardiac muscle is minimal. In contrast, Ins(1,4,5)P ₃ may play a pathological role in cardiac arrhythmogenesis by enhancing spontaneous Ca²⁺ ocsillations.     

Ryanodine modification of RyR1 retrogradely affects L-type Ca2+ channel gating in skeletal muscle

In skeletal muscle, there is bidirectional signalling between the L-type Ca²⁺ channel (1,4-dihydropyridine receptor; DHPR) and the type 1 ryanodine-sensitive Ca²⁺ release channel (RyR1) of the sarcoplasmic reticulum (SR). In the case of “orthograde signalling” (i.e., excitation-contraction coupling), the conformation of RyR1 is controlled by depolarization-induced conformational changes of the DHPR resulting in Ca²⁺ release from the SR. “Retrograde coupling” is manifested as enhanced L-type current. The nature of this retrograde signal, and its dependence on RyR1 conformation, are poorly understood. Here, we have examined L-type currents in normal myotubes after an exposure to ryanodine (200 μM, 1 h at 37°C) sufficient to lock RyR1 in a non-conducting, inactivated, conformational state. This treatment caused an increase in L-type current at less depolarized test potentials in comparison to myotubes similarly exposed to vehicle as a result of a ~5 mV hyperpolarizing shift in the voltage-dependence of activation. Charge movements of ryanodine-treated myotubes were also shifted to more hyperpolarizing potentials (~13 mV) relative to vehicle-treated myotubes. Enhancement of the L-type current by ryanodine was absent in dyspedic (RyR1 null) myotubes, indicating that ryanodine does not act directly on the DHPR. Our findings indicate that in retrograde signaling, the functional state of RyR1 influences conformational changes of the DHPR involved in activation of L-type current. This raises the possibility that physiological regulators of the conformational state of RyR1 (e.g., Ca²⁺, CaM, CaMK, redox potential) may also affect DHPR gating.     

Effects of thapsigargin and cyclopiazonic acid on sarcoplasmic reticulum Ca2+ uptake,spontaneous force oscillations and myofilament Ca2+ sensitivity in skinned rat ventricular trabeculae

Thapsigargin (TG) and cyclopiazonic acid (CPA) have been reported to be potent inhibitors of the sarcoplasmic reticulum (SR) Ca²⁺ uptake in isolated SR vesicles and cells. We have examined the effect of TG and CPA on (1) the Ca²⁺ uptake by the SR in saponin-skinned rat ventricular trabeculae, using the amplitude of the caffeine-induced contraction to estimate the Ca²⁺ content loaded into the SR, (2) the spontaneous Ca²⁺ oscillations at pCa 6.6 using force oscillation as the indicator, and (3) the myofilament Ca²⁺ sensitivity in Triton X-100-treated preparations. Inhibition of Ca²⁺ loading by TG and CPA increased with time of exposure to the inhibitor over 18–24 min. TG and CPA produced half inhibition of Ca²⁺ loading at 34.9 and 35.7 μM respectively, when 18–24 min were allowed for diffusion. The spontaneous force oscillations were more sensitive to the inhibitors: 10 μM TG and 30 μM CPA both abolished the oscillations in this time. The myofilament Ca²⁺ sensitivity was not affected by 10 and 300 μM TG or CPA. The results show that the concentrations of TG and CPA necessary to inhibit the SR Ca²⁺ uptake of skinned ventricular trabeculae are much higher than the reported values for single intact myocytes. One reason for this may be slow diffusion of the inhibitors into the multicellular trabecula preparation. Received: 28 July 1995/Received after revision: 11 December 1995/Accepted: 18 December 1995     

Evidence for the existence of inositol (1,4,5)-trisphosphate- and ryanodine-sensitive pools in bovine endothelial cells. Ca2+ releases in cells with different basal level of intracellular Ca2+

In single bovine aortic endothelial (BAE) cells pre-loaded with Fura-2, Ca²⁺ transients in a Ca²⁺-free medium have been revealed, which evidently reflects Ca²⁺ release from intracellular stores. In cells with different levels of resting basal cytoplasmic Ca²⁺ ([Ca²⁺]_i) from about 50 to 110 nM, a biphasic dependence of the Ca²⁺ transients on resting [Ca²⁺]_i was shown and spontaneous Ca²⁺ oscillations were observed. At a [Ca²⁺]_i level over 110 nM, a pronounced rise in Ca²⁺ transients occurred and only single transients were observed. Ryanodine (10 μM) produced a transient [Ca²⁺]_i elevation, suggesting the presence of ryanodine receptors in intracellular store membranes. The results imply that both inositol 1,4,5-trisphosphate-sensitive Ca²⁺ release (IICR) and Ca²⁺-sensitive Ca²⁺ release (CICR) take place in BAE cells. Only IICR seems to be sufficient for generating baseline Ca²⁺ oscillations in BAE cells, whereas the ATP-induced (5–100 μM) Ca²⁺ response involves the CICR set in motion by an oscillatory IICR of high frequency. The completion of both the spontaneous and ATP-induced Ca²⁺ transients was associated with a [Ca²⁺]_i decrease to a level below the initial resting [Ca²⁺]_i (undershoot). Its depth biphasically depended on the resting [Ca²⁺]_i from 50 to 110 nM, suggesting that the lack of a Ca²⁺ leak from inositol 1,4,5-trisphosphate-sensitive stores is responsible for the undershoot in this range. The Ca²⁺ leak is concluded to play a key role in the initiation and termination of regenerative IICR both in spontaneous oscillations and in ATP-induced transients. Received: 13 November 1995/Received after revision and accepted 27 March 1996     

[Ca2+]i elevation evoked by Ca2+ readdition to the medium after agonist-induced Ca2+ release can involve both IP 3-, and ryanodine-sensitive Ca2+ release

 Sustained Ca²⁺ elevation (”Ca²⁺ response”), caused by subsequent readdition of Ca²⁺ to the medium after application of adenosine 5’-triphosphate (ATP, 15 μM) in a Ca²⁺-free medium, was studied using single bovine aortic endothelial (BAE) cells. In cells in which the resting intracellular Ca²⁺ concentration ([Ca²⁺]_i) was between about 50 and 110 nM, a massive Ca²⁺ response occurred and consisted of phasic and sustained components, whereas cells with a resting [Ca²⁺]_i of over 110 nM displayed small plateau-like Ca²⁺ responses. An increase of internal store depletion resulted in loss of the phasic component. When the store was partly depleted, the dependence of the Ca²⁺ response amplitude on resting [Ca²⁺]_i was biphasic over the range of 50 to 110 nM. The greatest degree of store depletion was associated with small monophasic Ca²⁺ responses, the amplitudes of which were almost constant and in the same range as resting [Ca²⁺]_i. Ni²⁺, known to partly block Ca²⁺ entry, caused no change in the half-decay time of [Ca²⁺]_i down to the level of the sustained phase [57 ± 4 s in control and 54 ± 3 s (n = 13) in the presence of 10 mM Ni²⁺] when added at the peak of the phasic component of the Ca²⁺ response. However, it lowered the sustained phase of the Ca²⁺ response by 42%. When applied at the start of the readdition of Ca²⁺, Ni²⁺ blocked the phasic component of the Ca²⁺ response, there being a threefold decrease in the initial rate of [Ca²⁺]_i rise. In cells with a resting [Ca²⁺]_i of 75–80 nM, pre-treatment with ryanodine (10 μM) did not affect the peak amplitude of the Ca²⁺ response, but it did increase the level of the sustained component. In some cells, ryanodine caused an oscillatory Ca²⁺ response. In conclusion, partial depletion of the inositol 1,4,5-trisphosphate-(IP ₃-) sensitive store by a submaximal concentration of agonist (in Ca²⁺-free medium) was followed, on readdition of Ca²⁺, by Ca²⁺ entry, which appeared to trigger IP ₃-sensitive Ca²⁺ release (IICR) which, in turn, initiated Ca²⁺-sensitive Ca²⁺ release (CICR), thus resulting in a massive elevation of [Ca²⁺]_i. Received: 3 July 1996 / Received after revision and accepted: 9 September 1996     

Novel regulators of RyR Ca2+ release channels: insight into molecular changes in genetically-linked myopathies

There are many mutations in the ryanodine receptor (RyR) Ca²⁺ release channel that are implicated in skeletal muscle disorders and cardiac arrhythmias. More than 80 mutations in the skeletal RyR1 have been identified and linked to malignant hyperthermia, central core disease or multi-minicore disease, while more than 40 mutations in the cardiac RyR2 lead to ventricular arrhythmias and sudden cardiac death in patients with structurally normal hearts. These RyR mutations cause diverse changes in RyR activity which either excessively activate or block the channel in a manner that disrupts Ca²⁺ signalling in the muscle fibres. In a different myopathy, myotonic dystrophy (DM), a juvenile isoform of the skeletal RyR is preferentially expressed in adults. There are two regions of RyR1 that are variably spiced and developmentally regulated (ASI and ASII). The juvenile isoform (ASI (−)) is less active than the adult isoform (ASI(+)) and its over-expression in adults with DM may contribute to functional changes. Finally, mutations in an important regulator of the RyR, the Ca²⁺ binding protein calsequestrin (CSQ), have been linked to a disruption of Ca²⁺ homeostasis in cardiac myocytes that results in arrhythmias. We discuss evidence supporting the hypothesis that mutations in each of these situations alter protein/protein interactions within the RyR complex or between the RyR and its associated proteins. The disruption of these protein–protein interactions can lead either to excess Ca²⁺ release or reduced Ca²⁺ release and thus to abnormal Ca²⁺ homeostasis. Much of the evidence for disruption of protein–protein interactions has been provided by the actions of a group of novel RyR regulators, domain peptides with sequences that correspond to sequences within the RyR and which compete with the endogenous residues for their interaction sites.     

A novel Ca2+ binding protein associated with caldesmon in Ca2+-regulated smooth muscle thin filaments: evidence for a structurally altered form of calmodulin

Smooth muscle thin filaments are made up of actin, tropomyosin, the inhibitory protein caldesmon and a Ca²⁺-binding protein. Thin filament activation of myosin MgATPase is Ca²⁺-regulated but thin filaments assembled from smooth muscle actin, tropomyosin and caldesmon plus brain or aorta calmodulin are not Ca²⁺-regulated at 25°C/50 mM KCl. We isolated the Ca²⁺-binding protein (CaBP) from smooth muscle thin filaments by DEAE fast-flow chromatography in 6 M urea and phenyl sepharose chromatography using sheep aorta as our starting material. CaBP combines with smooth muscle actin, tropomyosin and caldesmon to reconstitute a normally regulated thin filament at 25°C/50 mM KCl. It reverses caldesmon inhibition at pCa5 under conditions where CaM is largely inactive, it binds to caldesmon when complexed with actin and tropomyosin rather than displacing it and it binds to caldesmon independently of [Ca²⁺]. Amino acid sequencing, and electrospray mass spectrometry show the CaBP is identical to CaM. Structural probes indicate it is different: calmodulin increases caldesmon tryptophan fluorescence but CaBP does not. The distribution of charged species in electrospray mass spectrometry and nozzle skimmer fragmentation patterns are different indicating a less stable N-terminal lobe for CaBP. Brief heating abolishes these special properties of the CaBP. Mass spectrometry in aqueous buffer showed no evidence for the presence of any covalent or non-covalently bound adduct. The only remaining conclusion is that CaBP is calmodulin locked in a metastable altered state.     

Altered Ca2+ sparks in aging skeletal and cardiac muscle

Ca²⁺ sparks are the fundamental units that comprise Ca²⁺-induced Ca²⁺ release (CICR) in striated muscle cells. In cardiac muscle, spontaneous Ca²⁺ sparks underlie the rhythmic CICR activity during heart contraction. In skeletal muscle, Ca²⁺ sparks remain quiescent during the resting state and are activated in a plastic fashion to accommodate various levels of stress. With aging, the plastic Ca²⁺ spark signal becomes static in skeletal muscle, whereas loss of CICR control leads to leaky Ca²⁺ spark activity in aged cardiomyocytes. Ca²⁺ spark responses reflect the integrated function of the intracellular Ca²⁺ regulatory machinery centered around the triad or dyad junctional complexes of striated muscles, which harbor the principal molecular players of excitation-contraction coupling. This review highlights the contribution of age-related modification of the Ca²⁺ release machinery and the effect of membrane structure and membrane cross-talk on the altered Ca²⁺ spark signaling during aging of striated muscles.     

Lobe-related concentration- and Ca2+-dependent interactions of calmodulin with C- and N-terminal tails of the CaV1.2 channel

This study examined the bindings of calmodulin (CaM) and its mutants with the C- and N-terminal tails of the voltage-gated Ca²⁺ channel Ca_V1.2 at different CaM and Ca²⁺ concentrations ([Ca²⁺]) by using the pull-down assay method to obtain basic information on the binding mode, including its concentration- and Ca²⁺-dependencies. Our data show that more than one CaM molecule could bind to the Ca_V1.2 C-terminal tail at high [Ca²⁺]. Additionally, the C-lobe of CaM is highly critical in sensing the change of [Ca²⁺] in its binding to the C-terminal tail of Ca_V1.2, and the binding between CaM and the N-terminal tail of Ca_V1.2 requires high [Ca²⁺]. Our data provide new details on the interactions between CaM and the Ca_V1.2 channel.     

ATP-induced Ca2+ signals in bronchial epithelial cells

 Ca²⁺-dependent Cl^– secretion in the respiratory tract occurs physiologically or under pathophysiological conditions when inflammatory mediators are released. The mechanism of intracellular Ca²⁺ release was investigated in the immortalized bronchial epithelial cell line 16HBE14o-. Experiments on both intact and permeabilized cells revealed that only inositol 1,4,5-trisphosphate (InsP ₃) receptors and not ryanodine receptors are involved in intracellular Ca²⁺ release. The expression pattern of the three InsP ₃ receptor isoforms was assessed both at the mRNA and at the protein level. The level of expression at the mRNA level was type 3 (92.5%) >> type 2 (5.4%) > type 1 (2.1%) and this rank order was also observed at the protein level. The ATP-induced Ca²⁺ signals in the intact cell, consisting of abortive Ca²⁺ spikes or fully developed [Ca²⁺] rises and intracellular Ca²⁺ waves, were indicative of positive feedback of Ca²⁺ on the InsP ₃ receptors. Low Ca²⁺ concentrations stimulated and high Ca²⁺ concentrations inhibited InsP ₃-induced Ca²⁺ release in permeabilized 16HBE14o- cells. We localized a cytosolic Ca²⁺-binding site between amino acid residues 2077 and 2101 in the type-2 InsP ₃ receptor and between amino acids 2030 and 2050 in the type-3 InsP ₃ receptor by expressing the respective parts of these receptors as glutathione S-transferase fusion proteins in bacteria. We conclude that the InsP ₃ receptor isoforms expressed in 16HBE14o- cells (mainly type-3 and type-2) are stimulated by Ca²⁺ and that this phenomenon contributes to the ATP-induced Ca²⁺ signals in intact 16HBE14o- cells. Recieved: 11 September 1997 / Received after revision: 2 January 1998 / Accepted: 21 January 1998     

An examination of the role of intracellular ATP in the activation of store-operated Ca2+ influx and Ca2+-dependent capacitance increases in rat basophilic leukaemia cells

 The role of ATP in both the activation of store-operated Ca²⁺ current I _CRAC and in Ca²⁺-dependent vesicular fusion was examined in a study of rat basophilic leukaemia (RBL) cells using the whole-cell patch-clamp technique. Fusion was monitored via changes in plasma membrane capacitance. Following a decrease in the levels of intracellular ATP, achieved using the mitochondrial poison antimycin and the ATP synthase inhibitor oligomycin, as well as a reduction of glycolysis by removal of external glucose, I _CRAC activated in a manner similar to control cells when stores are depleted by dialysis with a pipette solution containing either inositol 1,4,5-trisphosphate (InsP ₃) or ionomycin together with a high concentration of EGTA. Dialysis of cells for 150 s with the non-hydrolysable ATP analogue 5′-adenylylimidodiphosphate (AMP-PNP) (2 mM) in addition to the mitochondrial inhibitors also failed to prevent activation of I _CRAC following external application of ionomycin and thapsigargin, when compared with control recordings obtained with 2 mM ATP instead. Ca²⁺-dependent vesicular fusion was triggered by dialysing cells with 10 μM Ca²⁺ and guanosine-5′-O-(3-thiotriphosphate (GTP[γ-S]). The capacitance increase was unaffected by inhibition of glycolysis, mitochondrial inhibitors or dialysis with either AMP-PNP or adenosine 5′-O-(3-thiotriphosphate) (ATP[γ-S]) instead of ATP. We conclude that ATP hydrolysis does not seem to be necessary for the activation of I _CRAC or for the capacitance increases elicited by high concentrations of intracellular Ca²⁺. Received: 1 May 1998 / Received after revision: 16 July 1998 / Accepted: 16 July 1998     

Ca2+-dependent capacitance increases in rat basophilic leukemia cells following activation of store-operated Ca2+ entry and dialysis with high-Ca2+-containing intracellular solution

 Ca²⁺-dependent vesicular fusion was studied in single whole-cell patch-clamped rat basophilic leukemia (RBL) cells using the capacitance technique. Dialysis of the cells with 10 μM free Ca²⁺ and 300 μM guanosine 5′-O-(3-thiotriphosphate) (GTP[γ-S]) resulted in prominent capacitance increases. However, dialysis with either Ca²⁺ (225 nM to 10 μM) or GTP[γ-S] alone failed to induce a capacitance change. Under conditions of weak Ca²⁺ buffering (0.1 mM EGTA), activation of Ca²⁺-release-activated Ca²⁺ (CRAC) channels by dialysis with inositol 1,4,5-trisphosphate (InsP ₃) failed to induce a capacitance increase even in the presence of GTP[γ-S]. However, when Ca²⁺ATPases were inhibited by thapsigargin, InsP ₃ and GTP[γ-S] led to a pronounced elevation in membrane capacitance. This increase was dependent on a rise in intracellular Ca²⁺ because it was abolished when cells were dialysed with a high level of EGTA (10 mM) in the recording pipette. The increase was also dependent on Ca²⁺ influx because it was effectively suppressed when external Ca²⁺ was removed. Our results demonstrate that I _CRAC represents an important source of Ca²⁺ for triggering a secretory response. Received: 1 May 1998 / Received after revision: 15 June 1998 / Accepted: 2 July 1998     

Measurement of SR Ca2+ content in the presence of caffeine in permeabilised rat cardiac trabeculae

 This study was designed to measure the Ca²⁺ content of rat cardiac sarcoplasmic reticulum (SR) after equilibration with normal diastolic levels of Ca²⁺ (100 nM), in the absence and presence of caffeine. Measurements of [Ca²⁺] based on Fura-2 fluorescence were made from a limited bath volume (230 nl) containing individual saponin-permeabilised rat cardiac trabeculae. Injection of caffeine (5–40 mM) into this volume caused an initial release of Ca²⁺ from the SR, but within 30 s the SR was able to re-accumulate a significant proportion of the Ca²⁺. Ca²⁺ re-accumulation into the SR could be prevented by removal of ATP to inhibit the SR Ca²⁺ pump. Incubation of the preparation in an ATP-containing solution containing caffeine (5–40 mM) and 100 nM Ca²⁺ indicated that the SR’s ability to retain Ca²⁺ depends inversely on the dose of caffeine. The relative Ca²⁺ content of the SR after preincubation with caffeine was 86.7±3.5% at a caffeine concentration of 5 mM, 62.5±5.1% at 10 mM caffeine, 37.8±8.1% at 20 mM caffeine and 7.1±1.9% at 40 mM caffeine. Measurement of the SR Ca²⁺ release in the presence of different BAPTA concentrations was used to calculate (1) the Ca²⁺-binding capacity of the preparation (equivalent to 245±10 μM BAPTA) and (2) the Ca²⁺ content of the SR accessed by caffeine after equilibration with 100 nM Ca²⁺ (186±11 μmol/l cell volume or 5.6 mmol/l SR volume). Received: 9 June 1998 / Received after revision: 29 July 1998 / Accepted: 31 July 1998