Cytoplasmic Ca2+ does not inhibit the cardiac muscle sarcoplasmic reticulum ryanodine receptor Ca2+ channel,although Ca2+-induced Ca2+ inactivation of Ca2+ release is observed in native vesicles

Single channel properties of cardiac and fast-twitch skeletal muscle sarcoplasmic reticulum (SR) release channels were compared in a planar bilayer by fusing SR membranes in a Cs⁺-conducting medium. We found that the pharmacology, Cs⁺ conductance and selectivity to monovalent and divalent cations of the two channels were similar. The cardiac SR channel exhibited multiple kinetic states. The open and closed lifetimes were not altered from a range of 10^–7 to 10^–3 M Ca²⁺, but the proportion of closed and open states shifted to shorter closings and openings, respectively.However, while the single channel activity of the skeletal SR channel was activated and inactivated by micromolar and millimolar Ca²⁺, respectively, the cardiac SR channel remained activated in the presence of high [Ca²⁺]. In correlation to these studies, [³H]ryanodine binding by the receptors of the two channel receptors was inhibited by high [Ca²⁺] in skeletal but not in cardiac membranes in the presence of adenine nucleotides. There is, however, a minor inhibition of [³H]ryanodine binding of cardiac SR at millimolar Ca²⁺ in the absence of adenine nucleotides.When Ca²⁺-induced Ca²⁺ release was examined from preloaded native SR vesicles, the release rates followed a normal biphasic curve, with Ca²⁺-induced inactivation at high [Ca²⁺] for both cardiac and skeletal SR. Our data suggest that the molecular basis of regulation of the SR Ca²⁺ release channel in cardiac and skeletal muscle is different, and that the cardiac SR channel isoform lacks a Ca²⁺-inactivated site.This work was supported by research grants from the National Institutes of Health HL13870 and AR38970, and the Texas Affiliate of the American Heart Association, 91A-188. M. Fill was the recipient of an NIH fellowship AR01834.     

Glutamate receptor agonists evoked Ca2+-dependent and Ca2+-independent release of [3H]d-Aspartate from cultured chick retina cells

We studied the release of [³H]d-aspartate evoked by glutamate receptor agonists from monolayer cultures of chick retina cells, and found that activation of the glutamate receptors can evoke both Ca²⁺-dependent and Ca²⁺-independent release of [³H]d-aspartate. In Ca²⁺-free (no added Ca²⁺) Na⁺ medium, the agonists of the glutamate receptors induced the release of [³H]d-aspartate with the following rank order of potency: kainate>α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)∼N-methyl-d-aspartate (NMDA). In media containing 1 mM CaCl₂ the release of [³H]d-aspartate evoked by NMDA, kainate and AMPA was increased by about 112%, 20% and 39%, respectively, as compared to the release evoked by the same agonists in Ca²⁺-free medium. NMDA was the most potent agonist in stimulating the Ca²⁺-dependent release of [³H]d-aspartate, possibly by exocytosis, and AMPA was as potent as kainate. The Ca²⁺-dependent release of [³H]d-aspartate evoked by kainate was dependent on the influx of Ca²⁺ through the receptor associated channel, as well as through the N- (ω-Conotoxin GVIA-sensitive) and L- (nitrendipine-sensitive)type voltage-sensitive Ca²⁺ channels (VSCC). The exocytotic release of [³H]d-aspartate evoked by AMPA relied exclusively on Ca²⁺ entry through the L-type VSCC, whereas the effect of NMDA was partially mediated by the influx of Ca²⁺ through the receptor-associated channel, but not through L- or N-type VSCC. Thus, activation of these different glutamate receptors under physiological conditions is expected to cause the release of cytosolic and vesicular glutamate, and the routes of Ca²⁺ entry modulating vesicular release may be selectively recruited.     

Regulation of ryanodine receptors by sphingosylphosphorylcholine: Involvement of both calmodulin-dependent and -independent mechanisms

Sphingosylphosphorylcholine (SPC), a lipid mediator with putative second messenger functions, has been reported to regulate ryanodine receptors (RyRs), Ca²⁺ channels of the sarco/endoplasmic reticulum. RyRs are also regulated by the ubiquitous Ca²⁺ sensor calmodulin (CaM), and we have previously shown that SPC disrupts the complex of CaM and the peptide corresponding to the CaM-binding domain of the skeletal muscle Ca²⁺ release channel (RyR1). Here we report that SPC also displaces Ca²⁺-bound CaM from the intact RyR1, which we hypothesized might lead to channel activation by relieving the negative feedback Ca²⁺CaM exerts on the channel. We could not demonstrate such channel activation as we have found that SPC has a direct, CaM-independent inhibitory effect on channel activity, confirmed by both single channel measurements and [³H]ryanodine binding assays. In the presence of Ca²⁺CaM, however, the addition of SPC did not reduce [³H]ryanodine binding, which we could explain by assuming that the direct inhibitory action of the sphingolipid was negated by the simultaneous displacement of inhibitory Ca²⁺CaM. Additional experiments revealed that RyRs are unlikely to be responsible for SPC-elicited Ca²⁺ release from brain microsomes, and that SPC does not exert detergent-like effects on sarcoplasmic reticulum vesicles. We conclude that regulation of RyRs by SPC involves both CaM-dependent and -independent mechanisms, thus, the sphingolipid might play a physiological role in RyR regulation, but channel activation previously attributed to SPC is unlikely.     

Effects of 2,3-Butanedione 2-Monoxime on Ca2+ Release Channels (Ryanodine Receptors) of Cardiac and Skeletal Muscle

Single channel and [³H]ryanodine binding measurements were performed to test for a direct functional interaction between 2,3-butanedione 2-monoxime (BDM) and the skeletal and cardiac muscle sarcoplasmic reticulum Ca²⁺ release channels (ryanodine receptors). Single channel measurements were carried out in symmetric 0.25 m KCl media using the planar lipid bilayer method. BDM (1–10 mm) activated suboptimally Ca²⁺-activated (0.5–1 μm free Ca²⁺) single, purified and native cardiac and skeletal release channels in a concentration-dependent manner by increasing the number of channel events without a change of single channel conductances. BDM activated the two channel isoforms when added to either side of the bilayer. At a maximally activating cytosolic Ca²⁺ concentration of 20 μm, BDM was without effect on the cardiac channel, whereas it inhibited skeletal channel activities with IC₅₀≈ 2.5 mm. In agreement with single channel measurements, high-affinity [³H]ryanodine binding to the two channel isoforms was increased in a concentration-dependent manner at ≤1 μm Ca²⁺. BDM was without a noticeable effect at low (≤0.01 μm) Ca²⁺ concentrations. At 20 μm Ca²⁺, BDM inhibited the skeletal but not cardiac channel. These results suggest that BDM regulates the Ca²⁺ release channels from the sarcoplasmic reticulum of skeletal and cardiac muscle in a concentration, Ca²⁺ and tissue-dependent manner. Received: 31 December 1998/Revised: 9 March 1999     

N-type calcium channels are involved in the dopamine releasing effect of nicotine

Mouse striatum was incubated with [³H]dopamine ([³H]DA) and superfused with and the tritium efflux induced by nicotine, electrical stimulation, or simultaneous nicotine and electrical stimulation was measured, to characterize the role of different Ca²⁺ channels in the transmitter release. Nicotine stimulation and electrical stimulation exerted additive effects on tritium efflux. Separation of the released radioactivity on alumina columns indicated that nicotine or electrical stimulation increases the release of [³H]DA and that the outflow of³H-labeled metabolites was similar with the two different stimulation procedures. Removal of Ca²⁺ from the superfusate resulted in a marked reduction in the tritium release evoked by nicotine, whereas the electrical stimulation-evoked tritium release was completely dependent on external Ca²⁺. The L-and N-type calcium channel blockers omega-conotoxin GVIA and Cd²⁺ inhibited the tritium release from the striatum evoked by either nicotine or electrical stimulation, whereas the L-type and T-type channel blockers diltiazem and Ni²⁺ did not alter release of [³H]DA. We conclude that N-type voltage-sensitive Ca²⁺ channels participate in striatal dopamine release, and we speculate that nicotinic receptor-operated ion channels permeable to cations such as Ca²⁺ and N-type voltage-sensitive calcium channels may simultaneously open up, and they additively increase free intracellular Ca²⁺ concentration.     

Imperatoxin A Induces Subconductance States in Ca2+ Release Channels (Ryanodine Receptors) of Cardiac and Skeletal Muscle

Single-channel and [³H]ryanodine binding experiments were carried out to examine the effects of imperatoxin activator (IpTx_a), a 33 amino acid peptide isolated from the venom of the African scorpion Pandinus imperator, on rabbit skeletal and canine cardiac muscle Ca²⁺ release channels (CRCs). Single channel currents from purified CRCs incorporated into planar lipid bilayers were recorded in 250 mM KCl media. Addition of IpTx_a in nanomolar concentration to the cytosolic (cis) side, but not to the lumenal (trans) side, induced substates in both ryanodine receptor isoforms. The substates displayed a slightly rectifying current–voltage relationship. The chord conductance at −40 mV was ∼43% of the full conductance, whereas it was ∼28% at a holding potential of +40 mV. The substate formation by IpTx_a was voltage and concentration dependent. Analysis of voltage and concentration dependence and kinetics of substate formation suggested that IpTx_a reversibly binds to the CRC at a single site in the voltage drop across the channel. The rate constant for IpTx_a binding to the skeletal muscle CRC increased e-fold per +53 mV and the rate constant of dissociation decreased e-fold per +25 mV applied holding potential. The effective valence of the reaction leading to the substate was ∼1.5. The IpTx_a binding site was calculated to be located at ∼23% of the voltage drop from the cytosolic side. IpTx_a induced substates in the ryanodine-modified skeletal CRC and increased or reduced [³H]ryanodine binding to sarcoplasmic reticulum vesicles depending on the level of channel activation. These results suggest that IpTx_a induces subconductance states in skeletal and cardiac muscle Ca²⁺ release channels by binding to a single, cytosolically accessible site different from the ryanodine binding site.     

Effects of caffeine and raynodine on low pHi-induced changes in gap junction conductance and calcium concentration in crayfish septate axons

Summary Electrical uncoupling of crayfish septate axons with acidification has been shown to cause a substantial increase in [Ca²⁺]_i which closely matches in percent the increase in junctional resistance. To determine the origin of [Ca²⁺]_i increase, septate axons have been exposed either to drugs that influence Ca²⁺ release from internal stores, caffeine and ryanodine, or to treatments that affect Ca²⁺ entry. A large increase in junctional resistance and [Ca²⁺]_i maxima above controls resulted from addition of caffeine (10–30mm) to acetate solutions, while a substantial decrease in both parameters was observed when exposure to acetate-caffeine was preceded by caffeine pretreatment. In contrast, ryanodine (1–10 m) always caused a significant decrease in junctional resistance and [Ca²⁺]_i maxima when applied either together with acetate or both before and with acetate. Calcium channel blockers such as La³⁺, Cd²⁺ and nisoldipine had no effect, while an increase in the [Ca²⁺] of acetate solutions either decreased junctional resistance and [Ca²⁺]_i maxima or had no effect. The data suggest that cytoplasmic acidification causes an increase in [Ca²⁺]_i by releasing Ca²⁺ from caffeine and ryanodine-sensitive Ca²⁺ stores. The increase in [Ca²⁺]_i results in a decrease in gap junction conductance.     

The β1a Subunit of the Skeletal DHPR Binds to Skeletal RyR1 and Activates the Channel via Its 35-Residue C-Terminal Tail

Although it has been suggested that the C-terminal tail of the β_1a subunit of the skeletal dihyropyridine receptor (DHPR) may contribute to voltage-activated Ca²⁺ release in skeletal muscle by interacting with the skeletal ryanodine receptor (RyR1), a direct functional interaction between the two proteins has not been demonstrated previously. Such an interaction is reported here. A peptide with the sequence of the C-terminal 35 residues of β_1a bound to RyR1 in affinity chromatography. The full-length β_1a subunit and the C-terminal peptide increased [³H]ryanodine binding and RyR1 channel activity with an AC₅₀ of 450–600 pM under optimal conditions. The effect of the peptide was dependent on cytoplasmic Ca²⁺, ATP, and Mg²⁺ concentrations. There was no effect of the peptide when channel activity was very low as a result of Mg²⁺ inhibition or addition of 100 nM Ca²⁺ (without ATP). Maximum increases were seen with 1–10 μM Ca²⁺, in the absence of Mg²⁺ inhibition. A control peptide with the C-terminal 35 residues in a scrambled sequence did not bind to RyR1 or alter [³H]ryanodine binding or channel activity. This high-affinity in vitro functional interaction between the C-terminal 35 residues of the DHPR β_1a subunit and RyR1 may support an in vivo function of β_1a during voltage-activated Ca²⁺ release.     

The muscle ryanodine receptor and its intrinsic Ca2+ channel activity

In skeletal and cardiac muscle, contraction is initiated by the rapid release of Ca²⁺ ions from the intracellular membrane system, sarcoplasmic reticulum. Rapid-mixing vesicle ion flux and planar lipid bilayer-single-channel measurements have shown that Ca²⁺ release is mediated by a high-conductance, ligand-gated Ca²⁺ channel. Using the Ca²⁺ release-specific probe ryanodine, a 30 S protein complex composed of four polypeptides ofM _r 400,000 has been isolated. Reconstitution of the purified skeletal and cardiac muscle 30 S complexes into planar lipid bilayers induced single Ca²⁺ channel currents with conductance and gating kinetics similar to those of native Ca²⁺ release channels. Electron microscopy revealed structural similarity with the protein bridges (feet) that span the transverse-tubule-sarcoplasmic reticulum junction. These results suggest that striated muscle contains an intracellular Ca²⁺ release channel that is identical with the ryanodine receptor and the transverse-tubule-sarcoplasmic reticulum spanning feet structures.     

Modulation of Cardiac Sarcoplasmic Reticulum Calcium Release by Aenosine: A protein Kinase C- Dependent Pathway

We have already reported that A₃ adenosine receptor stimulation reduces [³H]-ryanodine binding and sarcoplasmic reticulum Ca²⁺ release in rat heart. In the present work we have investigated the transduction pathway responsible for this effect. Isolated rat hearts were perfused for 20 min in the presence of the following substances: 100 nM N⁶-(iodobenzyl)-adenosine-5′-N-methyluronamide (IB-MECA), an A₃ adenosine agonist; 10 μM U-73122, a phospholipase C inhibitor; 2 μM chelerythrine, a protein kinase C inhibitor. At the end of perfusion, the hearts were homogenized and [³H]-ryanodine binding was assayed. IB-MECA produced a significant decrease in ryanodine binding, which was abolished in the presence of chelerythrine but not in the presence of U-73122. RT-PCR experiments showed that ryanodine receptor gene expression was not affected by IB-MECA. In Western blot experiments, ryanodine receptor phosphorylation on serine 2809 was not modified after perfusion with IB-MECA. We conclude that modulation of SR Ca²⁺ release channel by IB-MECA is dependent on protein kinase C activation. However, in this model protein kinase C activation is not due to phospholipase C activation. In addition, changes in ryanodine receptor gene expression or direct phosphorylation of the ryanodine receptor on serine 2809 residue do not appear to occur.     

Effect of Subtype-Specific Ca2+-Antagonists and Ca2+-Free Media on the Field Stimulation-Evoked Release of ATP and [3H]Acetylcholine from Rat Habenula Slices

The involvement of different subtypes of voltage-sensitive Ca²⁺ channels in the initiation of field stimulation-induced endogenous adenosine triphosphate (ATP) and [³H]acetylcholine ([³H]ACh) release was investigated in the superfused rat habenula slices. ATP, measured by the luciferin-luciferase assay, and [³H]ACh were released simultaneously from the tissue in response to low frequency electrical stimulation (2 Hz, 2.5 msec, 360 shocks). The N-type Ca²⁺ channel blocker -conotoxin GVIA (-CgTX, 0.01–1 M) reduced the stimulation-evoked release of ATP and [³H]ACh in a dose-dependent manner. Similarly, the P-type Ca²⁺ channel antagonist -agatoxin IVA (-Aga IVA) (0.05 M) and the inorganic Ca²⁺ channel blocker Cd²⁺ (0.2 mM) inhibited the outflow of both transmitters, while Ni²⁺ (0.1 mM) was without significant effect. A high correlation was observed between the percent inhibition of ATP release and percent inhibition of ACh release caused by the different Ca²⁺ antagonists. Long-term perfusion (i.e., 90 min) with Ca²⁺ free solution inhibited the evoked-release of ATP and [³H]ACh. In contrast, perfusion of slices with the same media for a shorter time (i.e., 20 min) did not reduce the release of [³H]ACh and ATP but even increased the evoked-release of ATP about fourfold. The breakdown of extracellular ATP was not blocked under low [Ca²⁺]₀ condition, measured by the creatine phosphokinase assay and HPLC-UV technique. Application of extra- or intracellular Ca²⁺ chelators, and dipyridamole (2 M), the nucleoside transporter inhibitor, did not reduce the excess release of ATP after short-term perfusion with Ca²⁺-free media. Tetrodotoxin (TTX, 1 M), while inhibiting the majority of ATP release under normal conditions, was also unable to reduce release under low [Ca²⁺]₀ conditions. In summary, we showed that both N- and P-type Ca²⁺ channels are involved in the initiation of electrical stimulation-evoked release of ATP and [³H]ACh in the rat habenula under normal extracellular calcium concentration. Under low [Ca²⁺]₀ conditions an additional release of ATP occurs, which is not associated with action potential propagation.     

Ca-Calmodulin Increases RyR2 Open Probability yet Reduces Ryanoid Association with RyR2

We have shown that physiological levels of Ca²⁺-calmodulin (Ca²⁺CaM; 50-100 nM) activate cardiac ryanodine receptors (RyR2) incorporated into bilayers and increase the frequency of Ca²⁺ sparks and waves in cardiac cells. In contrast, it is well known that Ca²⁺CaM inhibits [³H]ryanodine binding to cardiac sarcoplasmic reticulum. Since the [³H]ryanodine binding technique does not reflect the effects of Ca²⁺CaM on RyR2 open probability (Po), we have investigated, using the reversible ryanoid, ryanodol, whether Ca²⁺CaM can directly influence the binding of ryanoids to single RyR2 channels independently of Po. We demonstrate that Ca²⁺CaM reduces the rate of ryanodol association to RyR2 without affecting the rate of dissociation. We also find that ryanodol-bound channels fluctuate between at least two distinct subconductance states, M₁ and M₂, in a voltage-dependent manner. Ca²⁺CaM significantly alters the equilibrium between these two states. The results suggest that Ca²⁺CaM binding to RyR2 causes a conformation change to regions of the channel that include the ryanoid binding site, thereby leading to a decrease in ryanoid association rate and modulation of gating within the ryanoid/RyR2 bound state. Our data provide a possible explanation for why the effects of Ca²⁺CaM at the single-channel level are not mirrored by [³H]ryanodine binding studies.     

Reconstitution and regulation of cation-selective channels from cardiac sarcoplasmic reticulum

In order to study the conductances of the Sarcoplasmic Reticulum (SR) membrane, microsomal fractions from cardiac SR were isolated by differential and sucrose gradient centrifugations and fused into planar lipid bilayers (PLB) made of phospholipids. Using either KCl or K-gluconate solutions, a large conducting K+ selective channel was characterized by its ohmic conductance (152 pS in 150 mM K⁺), and the presence of short and long lasting subconducting states. Its open probability P_o increased with depolarizing voltages, thus supporting the idea that this channel might allow counter-charge movements of monovalent cations during rapid SR Ca²⁺ release. An heterogeneity in the kinetic behavior of this channel would suggest that the cardiac SR K⁺ channels might be regulated by cytoplasmic, luminal, or intra SR membrane biochemical mechanisms. Since the behavior was not modified by variations of [Ca²⁺] nor by the addition of soluble metabolites such as ATP, GTP, cAMP, cGMP, nor by phosphorylation conditions on both sides of the PLB, a specific interaction with a SR membrane component is postulated. Another cation selective channel was studied in asymmetric Ca²⁺, Ba²⁺ or Mg²⁺-HEPES buffers. This channel displayed large conductance values for the above divalent cations 90, 100, and 40 pS, respectively. This channel was activated by µM Ca²⁺ while its Ca²⁺ sensitivity was potentiated by millimolar ATP. However Mg²⁺ and calmodulin modulated its gating behavior. Ca²⁺ releasing drugs such as caffeine and ryanodine increased its P_o. All these features are characteristics of the SR Ca²⁺ release channel. The ryanodine receptor which has been purified and reconstituted into PLB, may form a cation selective pathway. This channel displays all the regulatory sites of the native cardiac SR Ca²⁺ release channel. However, when NA was used as charge carrier, multiple subconducting states were observed. In conclusion, the reconstitution experiments have yield a great deal of informations about the biochemical and biophysical events that may regulated the ionic flux across the SR membrane.     

Endothelin-1 induces intracellular [Ca2+] increase via Ca2+ influx through the L-type Ca2+ channel, Ca2+-induced Ca2+ release and a pathway involving ETA receptors, PKC, PKA and AT1 receptors in cardiomyocytes

Using fura-2-acetoxymethyl ester (AM) fluorescence imaging and patch clamp techniques, we found that endothelin-1 (ET-1) significantly elevated the intracellular calcium level ([Ca²⁺]_i) in a dose-dependent manner and activated the L-type Ca²⁺ channel in cardiomyocytes isolated from rats. The effect of ET-1 on [Ca²⁺]_i elevation was abolished in the presence of the ET_A receptor blocker BQ123, but was not affected by the ET_B receptor blocker BQ788. ET-1-induced an increase in [Ca²⁺]_i, which was inhibited 46.7% by pretreatment with a high concentration of ryanodine (10 μmol/L), a blocker of the ryanodine receptor. The ET-1-induced [Ca²⁺]_i increase was also inhibited by the inhibitors of protein kinase A (PKA), protein kinase C (PKC) and angiotensin type 1 receptor (AT1 receptor). We found that ET-1 induced an enhancement of the amplitude of the whole cell L-type Ca²⁺ channel current and an increase of open-state probability (NPo) of an L-type single Ca²⁺ channel. BQ123 completely blocked the ET-1-induced increase in calcium channel open-state probability. In this study we demonstrated that ET-1 regulates calcium overload through a series of mechanisms that include L-type Ca²⁺ channel activation and Ca²⁺-induced Ca²⁺ release (CICR). ETA receptors, PKC, PKA and AT1 receptors may also contribute to this pathway. Supported by the National Natural Science Foundation of China (Grant No. 200830870910).     

The action of carboxyl modifying reagents on the ryanodine receptor/Ca2+ release channel of skeletal muscle sarcoplasmic reticulum

Summary

In this work we show that ryanodine binding to junctional sarcoplasmic reticulum (SR) membranes or purified ryanodine receptor (RyR) is inhibited in a time — and concentration-dependent fashion by prior treatment with the carboxyl reagent dicyclohexylcarbodiimide (DCCD). Exposure of the membrane-bound RyR to the water soluble carboxyl reagents 1-ethyl-3 (3-(dimethylamino) propyl carbodiimide (EDC) or N-ethyl-pheny-lisoxazolium-3 -sulfonate (WRK) only slightly affects their ryanodine binding capacity. The amphipathic reagent N-ethoxy cabonyl-2-ethoxy-1, 2-dihydroquinaline (EEDQ) inhibited ryanodine binding at relatively high concentrations. DCCD-modifica-tion of the SR decreased the binding affinities of the RyR for ryanodine and Ca²⁺ by about 3- and 18-fold, respectively.

The single channel activity of SR membranes modified with DCCD and then incorporated into planar lipid bilayers is very low (5–8%) in comparison to control membranes. Application of DCCD to either the myoplasmic (c/s) or luminal (trans) side of the reconstituted unmodified channels resulted in complete inhibition of their single channel activities. Similar results were obtained with the water soluble reagent WRK applied to the myoplasmic, but not to the luminal side. The DCCD-modified non-active channel is re-activated by addition of ryanodine in the presence of 250_üM Ca²⁺ and is stabilized in a sub-conductance state. With caffeine, ryanodine re-activated the channel in the presence of 100_üM of Ca^2+. The results suggest that a carboxyl residue(s) in the RyR is involved either in the binding of Ca²⁺, or in conformational changes that are produced by Ca²⁺ binding, and are required for the binding of ryanodine and the opening of the Ca²⁺ release channel.     

The actin cytoskeleton differentially regulates NG115-401L cell ryanodine receptor and inositol 1,4,5-trisphosphate receptor induced calcium signaling pathways

Regulation of bi-directional communication between intracellular Ca²⁺ pools and surface Ca²⁺ channels remains incompletely characterized. We report Ca²⁺ release mediated by inositol 1,4,5-trisphosphate receptor (IP₃R) and ryanodine receptor (RyR) pathways is diminished under actin cytoskeleton disruption in NG115-401L (401L) neuronal cells, yet despite truncated Ca²⁺ release, Ca²⁺ influx was not significantly altered in these experiments. However, disruption of cortical actin networks completely abolished IP₃R induced Ca²⁺ release, whereas RyR-mediated Ca²⁺ release was preserved, albeit attenuated. Moreover, cortical actin disruption completely abolished IP₃R and RyR linked Ca²⁺ influx even though Ca²⁺ pool sensitivities were different. These findings suggest discrete Ca²⁺ store/Ca²⁺ channel coupling mechanisms in the IP₃R and RyR pathways as revealed by the differential sensitivity to actin perturbation.     

Characterisation and Distribution of Inositol Polyphosphate and Ryanodine Receptors in the Rat Brain

Abstract: The regional distribution of inositol 1,4,5-trisphosphate (InsP₃), inositol 1,3,4,5-tetrakisphosphate (InsP₄), and ryanodine binding sites has been characterised and compared in the rat brain using radioligand binding assays. Cortical [³H]InsP₃ binding indicated similar positional and stereospecificity as observed in other tissues, with 100-fold selectivity for lnsP₃ over InsP₄. Similarly, high-affinity [³²P]InsP₄ binding also showed a high degree of positional specificity, with a 1,000-fold selectivity for InsP₄ over InsP₃. Initial characterisation of [³H]ryanodine binding to cortical membranes demonstrated that specific binding was highly dependent on high salt and micromolar Ca²⁺ concentrations and inhibited by Ca²⁺ levels of >1 mM. [³H]-Ryanodine binding was also enhanced by β,γ-methylene-adenosine 5′-trisphosphate and caffeine and inhibited by magnesium and ruthenium red (K_i= 0.81 μM). However, dantrolene (300 μM) was ineffective on the binding. Therefore, although the results indicate a greater similarity to the binding properties of the Ca²⁺-induced Ca²⁺ release channel isoform present in skeletal, rather than cardiac, muscle, it does not appear to be identical. Detailed binding analysis of ryanodine and polyphosphate sites, with the exception of ruthenium red, indicated no interaction between binding sites. Ruthenium red markedly enhanced the binding of both [³H]InsP₃ and [³²P]InsP₄, an effect most probably due to nonspecific complex formation. Regional binding of InP₃, InsP₄, and ryanodine in the rat brain was of similar affinity for each ligand in each area, but the density profile for each ligand was clearly different. The highest density of InsP₃ sites was in the cerebellum, whereas the highest density of ryanodine sites was in the hippocampus. High-affinity InsP₄ sites showed less regional diversity, with highest densities in the cerebellum, cortex, and hippocampus. However, in each area studied the density of sites followed the order InsP₃ > InsP₄ > ryanodine.     

Biochemical characterization of the Ca2+ release channel of skeletal and cardiac sarcoplasmic reticulum

Summary Rapid mixing-vesicle ion flux and planar lipid bilayer-single channel measurements have shown that a high-conductance, ligand-gated Ca²⁺ release channel is present in heavy, junctional-derived membrane fractions of skeletal and cardiac muscle sarcoplasmic reticulum. Using the release channel-specific probe, ryanodine, a 30S protein complex composed of polypeptides of M_r 400 000 has been isolated from cardiac and skeletal muscle. Reconstitution of the complex into planar lipid bilayers has revealed a Ca²⁺ conductance with properties characteristic of the native Ca²⁺ release channel.     

Cytoplasmic Ca2+ inhibits the ryanodine receptor from cardiac muscle

Ca²⁺-dependent inhibition of native and isolated ryanodine receptor (RyR) calcium release channels from sheep heart and rabbit skeletal muscle was investigated using the lipid bilayer technique. We found that cytoplasmic Ca²⁺ inhibited cardiac RyRs with an average K _m = 15 mm, skeletal RyRs with K _m = 0.7 mm and with Hill coefficients of 2 in both isoforms. This is consistent with measurements of Ca²⁺ release from the sarcoplasmic reticulum (SR) in skinned fibers and with [³H]-ryanodine binding to SR vesicles, but is contrary to previous bilayer studies which were unable to demonstrate Ca²⁺-inhibition in cardiac RyRs (Chu, Fill, Stefani &; Entman (1993) J. Membrane Biol. 135, 49–59). Ryanodine prevented Ca²⁺ from inhibiting either cardiac or skeletal RyRs. Ca²⁺-inhibition in cardiac RyRs appeared to be the most fragile characteristic of channel function, being irreversibly disrupted by 500 mm Cs⁺, but not by 500 mm K⁺, in the cis bath or by solublization with the detergent CHAPS. These treatments had no effect on channel regulation by AMP-PNP, caffeine, ryanodine, ruthenium red, or Ca²⁺-activation. Ca²⁺-inhibition in skeletal RyRs was retained in the presence of 500 mm Cs⁺. Our results provide an explanation for previous findings in which cardiac RyRs in bilayers with 250 mm Cs⁺ in the solutions fail to demonstrate Ca²⁺-inhibition, while Ca²⁺-inhibition of Ca²⁺ release is observed in vesicle studies where K⁺ is the major cation. A comparison of open and closed probability distributions from individual RyRs suggested that the same gating mechanism mediates Ca²⁺-inhibition in skeletal RyRs and cardiac RyRs, with different Ca²⁺ affinities for inhibition. We conclude that differences in the Ca²⁺-inhibition in cardiac and skeletal channels depends on their Ca²⁺ binding properties.     

Longitudinal smooth muscle of the mammalian intestine

Ca²⁺ mobilization in muscle cells from the circular muscle layer of the mammalian intestine is mediated by IP₃-dependent Ca²⁺ release. Ca²⁺ mobilization in muscle from the adjacent longitudinal muscle layer involves a distinct, phosphoinositide-independent pathway. Receptors for contractile agonists in longitudinal muscle cells are coupled via a pertussis toxinsensitive G protein to activation of PLA₂ and formation of arachidonic acid (AA). The latter activates Cl⁻ channels resulting in depolarization of the plasma membrane and opening of voltage-sensitive Ca²⁺ channels. Ca²⁺ influx via these channels induces Ca²⁺ release by activating sarcoplasmic ryanodine receptor/Ca²⁺ channels. The increase in [Ca²⁺]_i activates membrane-bound ADP ribosyl cyclase, and the resultant formation of cADPR enhances Ca²⁺-induced Ca²⁺ release.