Phenytoin Reduces Activity of Cardiac Ryanodine Receptor 2; A Potential Mechanism for Its Cardioprotective Action.

Phenytoin is a hydantoin derivative that is used clinically for the treatment of epilepsy and has been reported to have antiarrhythmic actions on the heart. In a failing heart, the elevated diastolic Ca2+ leak from the sarcoplasmic reticulum can be normalized by the cardiac ryanodine receptor 2 (RyR2) inhibitor, dantrolene, without inhibiting Ca2+ release during systole or affecting Ca2+ release in normal healthy hearts. Unfortunately, dantrolene is hepatotoxic and unsuitable for chronic long-term administration. Because phenytoin and dantrolene belong to the hydantoin class of compounds, we test the hypothesis that dantrolene and phenytoin have similar inhibitory effects on RyR2 using a single-channel recording of RyR2 activity in artificial lipid bilayers. Phenytoin produced a reversible inhibition of RyR2 channels from sheep and human failing hearts. It followed a hyperbolic dose response with maximal inhibition of ∼50%, Hill coefficient ∼1, and IC50 ranging from 10 to 20 µM. It caused inhibition at diastolic cytoplasmic [Ca2+] but not at Ca2+ levels in the dyadic cleft during systole. Notably, phenytoin inhibits RyR2 from failing human heart but not from healthy heart, indicating that phenytoin may selectively target defective RyR2 channels in humans. We conclude that phenytoin could effectively inhibit RyR2-mediated release of Ca2+ in a manner paralleling that of dantrolene. Moreover, the IC50 of phenytoin in RyR2 is at least threefold lower than for other ion channels and clinically used serum levels, pointing to phenytoin as a more human-safe alternative to dantrolene for therapies against heart failure and cardiac arrythmias. SIGNIFICANCE STATEMENT: We show that phenytoin, a Na channel blocker used clinically for treatment of epilepsy, is a diastolic inhibitor of cardiac calcium release channels [cardiac ryanodine receptor 2 (RyR2)] at doses threefold lower than its current therapeutic levels. Phenytoin inhibits RyR2 from failing human heart and not from healthy heart, indicating that phenytoin may selectively target defective RyR2 channels in humans and pointing to phenytoin as a more human-safe alternative to dantrolene for therapies against heart failure and cardiac arrhythmias.

Ryanodine receptor modification and regulation by intracellular Ca2+ and Mg2+ in healthy and failing human hearts.

RATIONALE: Heart failure is a multimodal disorder, of which disrupted Ca2+ homeostasis is a hallmark. Central to Ca2+ homeostasis is the major cardiac Ca2+ release channel - the ryanodine receptor (RyR2) - whose activity is influenced by associated proteins, covalent modification and by Ca2+ and Mg2+. That RyR2 is remodelled and its function disturbed in heart failure is well recognized, but poorly understood. OBJECTIVE: To assess Ca2+ and Mg2+ regulation of RyR2 from left ventricles of healthy, cystic fibrosis and failing hearts, and to correlate these functional changes with RyR2 modifications and remodelling. METHODS AND RESULTS: The function of RyR2 from left ventricular samples was assessed using lipid bilayer single-channel measurements, whilst RyR2 modification and protein:protein interactions were determined using Western Blots and co-immunoprecipitation. In all failing hearts there was an increase in RyR2 activity at end-diastolic cytoplasmic Ca2+ (100nM), a decreased cytoplasmic [Ca2+] required for half maximal activation (Ka) and a decrease in inhibition by cytoplasmic Mg2+. This was accompanied by significant hyperphosphorylation of RyR2 S2808 and S2814, reduced free thiol content and a reduced interaction with FKBP12.0 and FKBP12.6. Either dephosphorylation of RyR2 using PP1 or thiol reduction using DTT eliminated any significant difference in the activity of RyR2 from healthy and failing hearts. We also report a subgroup of RyR2 in failing hearts that were not responsive to regulation by intracellular Ca2+ or Mg2+. CONCLUSION: Despite different aetiologies, disrupted RyR2 Ca2+ sensitivity and biochemical modification of the channel are common constituents of failing heart RyR2 and may underlie the pathological disturbances in intracellular Ca2+ signalling.

Mechanisms of SR calcium release in healthy and failing human hearts.

Normal heart contraction and rhythm relies on the proper flow of calcium ions (Ca2+) into cardiac cells and between their intracellular organelles, and any disruption can lead to arrhythmia and sudden cardiac death. Electrical excitation of the surface membrane activates voltage-dependent L-type Ca2+ channels to open and allow Ca2+ to enter the cytoplasm. The subsequent increase in cytoplasmic Ca2+ concentration activates calcium release channels (RyR2) located at specialised Ca2+ release sites in the sarcoplasmic reticulum (SR), which serves as an intracellular Ca2+ store. Animal models have provided valuable insights into how intracellular Ca2+ transport mechanisms are altered in human heart failure. The aim of this review is to examine how Ca2+ release sites are remodelled in heart failure and how this affects intracellular Ca2+ transport with an emphasis on Ca2+ release mechanisms in the SR. Current knowledge on how heart failure alters the regulation of RyR2 by Ca2+ and Mg2+ and how these mechanisms control the activity of RyR2 in the confines of the Ca2+ release sites is reviewed.

Multiple modes of ryanodine receptor 2 inhibition by flecainide.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) causes sudden cardiac death due to mutations in cardiac ryanodine receptors (RyR2), calsequestrin, or calmodulin. Flecainide, a class I antiarrhythmic drug, inhibits Na(+) and RyR2 channels and prevents CPVT. The purpose of this study is to identify inhibitory mechanisms of flecainide on RyR2. RyR2 were isolated from sheep heart, incorporated into lipid bilayers, and investigated by single-channel recording under various activating conditions, including the presence of cytoplasmic ATP (2 mM) and a range of cytoplasmic [Ca(2+)], [Mg(2+)], pH, and [caffeine]. Flecainide applied to either the cytoplasmic or luminal sides of the membrane inhibited RyR2 by two distinct modes: 1) a fast block consisting of brief substate and closed events with a mean duration of ∼1 ms, and 2) a slow block consisting of closed events with a mean duration of ∼1 second. Both inhibition modes were alleviated by increasing cytoplasmic pH from 7.4 to 9.5 but were unaffected by luminal pH. The slow block was potentiated in RyR2 channels that had relatively low open probability, whereas the fast block was unaffected by RyR2 activation. These results show that these two modes are independent mechanisms for RyR2 inhibition, both having a cytoplasmic site of action. The slow mode is a closed-channel block, whereas the fast mode blocks RyR2 in the open state. At diastolic cytoplasmic [Ca(2+)] (100 nM), flecainide possesses an additional inhibitory mechanism that reduces RyR2 burst duration. Hence, multiple modes of action underlie RyR2 inhibition by flecainide.

Control of sarcoplasmic reticulum Ca2+ release by stochastic RyR gating within a 3D model of the cardiac dyad and importance of induction decay for CICR termination.

The factors responsible for the regulation of regenerative calcium-induced calcium release (CICR) during Ca(2+) spark evolution remain unclear. Cardiac ryanodine receptor (RyR) gating in rats and sheep was recorded at physiological Ca(2+), Mg(2+), and ATP levels and incorporated into a 3D model of the cardiac dyad, which reproduced the time course of Ca(2+) sparks, Ca(2+) blinks, and Ca(2+) spark restitution. The termination of CICR by induction decay in the model principally arose from the steep Ca(2+) dependence of RyR closed time, with the measured sarcoplasmic reticulum (SR) lumen Ca(2+) dependence of RyR gating making almost no contribution. The start of CICR termination was strongly dependent on the extent of local depletion of junctional SR Ca(2+), as well as the time course of local Ca(2+) gradients within the junctional space. Reducing the dimensions of the dyad junction reduced Ca(2+) spark amplitude by reducing the strength of regenerative feedback within CICR. A refractory period for Ca(2+) spark initiation and subsequent Ca(2+) spark amplitude restitution arose from 1), the extent to which the regenerative phase of CICR can be supported by the partially depleted junctional SR, and 2), the availability of releasable Ca(2+) in the junctional SR. The physical organization of RyRs within the junctional space had minimal effects on Ca(2+) spark amplitude when more than nine RyRs were present. Spark amplitude had a nonlinear dependence on RyR single-channel Ca(2+) flux, and was approximately halved by reducing the flux from 0.6 to 0.2 pA. Although rat and sheep RyRs had quite different Ca(2+) sensitivities, Ca(2+) spark amplitude was hardly affected. This suggests that moderate changes in RyR gating by second-messenger systems will principally alter the spatiotemporal properties of SR release, with smaller effects on the amount released.

Termination of calcium-induced calcium release by induction decay: an emergent property of stochastic channel gating and molecular scale architecture.

Calcium-induced calcium release (CICR) is an inherently regenerative process due to the Ca(2+)-dependent gating of ryanodine receptors (RyRs) in the sarco/endoplasmic reticulum (SR) and is critical for cardiac excitation-contraction coupling. This process is seen as Ca(2+) sparks, which reflect the concerted gating of groups of RyRs in the dyad, a specialised junctional signalling domain between the SR and surface membrane. However, the mechanism(s) responsible for the termination of regenerative CICR during the evolution of Ca(2+) sparks remain uncertain. Rat cardiac RyR gating was recorded at physiological Ca(2+), Mg(2+) and ATP levels and incorporated into a 3D model of the cardiac dyad which reproduced the time-course of Ca(2+) sparks, Ca(2+) blinks and Ca(2+) spark restitution. Model CICR termination was robust, relatively insensitive to the number of dyadic RyRs and automatic. This emergent behaviour arose from the rapid development and dissolution of nanoscopic Ca(2+) gradients within the dyad. These simulations show that CICR does not require intrinsic inactivation or SR calcium sensing mechanisms for stability and cessation of regeneration that arises from local control at the molecular scale via a process we call 'induction decay'.

Regulation of ryanodine receptors from skeletal and cardiac muscle during rest and excitation.

1. In muscle, intracellular calcium concentration, hence skeletal muscle force and cardiac output, is regulated by uptake and release of calcium from the sarcoplasmic reticulum (SR). The ryanodine receptor (RyR) forms the calcium release channel in the SR. 2. Calcium release through RyRs is modulated by a wide variety of endogenous molecules, including small diffusible ligands such as ATP, Ca2+ and Mg2+. The regulation of RyR channels by ATP, Ca2+ and Mg2+ is a complex interplay of several regulatory mechanisms, which are still being unravelled. Consequently, it is not clearly known how RyRs are regulated in resting muscle and during contraction. 3. The present paper reviews factors controlling the activity of RyRs in skeletal and cardiac muscle with an emphasis on mechanistic insights derived from single channel recording methods. 4. In addition, the nature of dihydropyridine receptor (DHPR) control of RyRs in skeletal muscle derived from experiments with peptide fragments of the DHPR II-III loop is reviewed. 5. Finally, recent experiments on coupled RyRs in lipid bilayers and their potential for resolving the elusive mechanisms controlling calcium release during cardiac contraction are discussed.

Calsequestrin and the calcium release channel of skeletal and cardiac muscle.

Calsequestrin is by far the most abundant Ca(2+)-binding protein in the sarcoplasmic reticulum (SR) of skeletal and cardiac muscle. It allows the Ca2+ required for contraction to be stored at total concentrations of up to 20mM, while the free Ca2+ concentration remains at approximately 1mM. This storage capacity confers upon muscle the ability to contract frequently with minimal run-down in tension. Calsequestrin is highly acidic, containing up to 50 Ca(2+)-binding sites, which are formed simply by clustering of two or more acidic residues. The Kd for Ca2+ binding is between 1 and 100 microM, depending on the isoform, species and the presence of other cations. Calsequestrin monomers have a molecular mass of approximately 40 kDa and contain approximately 400 residues. The monomer contains three domains each with a compact alpha-helical/beta-sheet thioredoxin fold which is stable in the presence of Ca2+. The protein polymerises when Ca2+ concentrations approach 1mM. The polymer is anchored at one end to ryanodine receptor (RyR) Ca2+ release channels either via the intrinsic membrane proteins triadin and junctin or by binding directly to the RyR. It is becoming clear that calsequestrin has several functions in the lumen of the SR in addition to its well-recognised role as a Ca2+ buffer. Firstly, it is a luminal regulator of RyR activity. When triadin and junctin are present, calsequestrin maximally inhibits the Ca2+ release channel when the free Ca2+ concentration in the SR lumen is 1mM. The inhibition is relieved when the Ca2+ concentration alters, either because of small changes in the conformation of calsequestrin or its dissociation from the junctional face membrane. These changes in calsequestrin's association with the RyR amplify the direct effects of luminal Ca2+ concentration on RyR activity. In addition, calsequestrin activates purified RyRs lacking triadin and junctin. Further roles for calsequestrin are indicated by the kinase activity of the protein, its thioredoxin-like structure and its influence over store operated Ca2+ entry. Clearly, calsequestrin plays a major role in calcium homeostasis that extends well beyond its ability to buffer Ca2+ ions.

Suppression of calcium sparks in rat ventricular myocytes and direct inhibition of sheep cardiac RyR channels by EPA, DHA and oleic acid.

The anti-arrhythmic effects of long-chain polyunsaturated fatty acids (PUFAs) may be related to their ability to alter calcium handling in cardiac myocytes. We investigated the effect of eicosapentanoic acid (EPA) and docosahexaenoic acid (DHA) on calcium sparks in rat cardiac myocytes and the effects of these PUFAs and the monounsaturated oleic acid on cardiac calcium release channels (RyRs). Visualization of subcellular calcium concentrations in single rat ventricular myocytes showed that intensity of calcium sparks was reduced in the presence of EPA and DHA (15 micro M). It was also found that calcium sparks decayed more quickly in the presence of EPA but not DHA. Sarcoplasmic vesicles containing RyRs were prepared from sheep hearts and RyR activity was determined by either [(3)H]ryanodine binding or by single-channel recording. Bilayers were formed from phosphatidylethanolamine and phosphatidylcholine dissolved in either n-decane or n-tetradecane. EPA inhibited [(3)H]ryanodine binding to RyRs in SR vesicles with K(I) = 40 micro M. Poly- and mono-unsaturated free fatty acids inhibited RyR activity in lipid bilayers. EPA (cytosolic or luminal) inhibited RyRs with K(I) =32 micro M and Hill coefficient, n(1) = 3.8. Inhibition was independent of the n-alkane solvent and whether RyRs were activated by ATP or Ca(2+). DHA and oleic acid also inhibited RyRs, suggesting that free fatty acids generally inhibit RyRs at micromolar concentrations.

Interactions between dihydropyridine receptors and ryanodine receptors in striated muscle.

Excitation-contraction coupling in both skeletal and cardiac muscle depends on structural and functional interactions between the voltage-sensing dihydropyridine receptor L-type Ca(2+) channels in the surface/transverse tubular membrane and ryanodine receptor Ca(2+) release channels in the sarcoplasmic reticulum membrane. The channels are targeted to either side of a narrow junctional gap that separates the external and internal membrane systems and are arranged so that bi-directional structural and functional coupling can occur between the proteins. There is strong evidence for a physical interaction between the two types of channel protein in skeletal muscle. This evidence is derived from studies of excitation-contraction coupling in intact myocytes and from experiments in isolated systems where fragments of the dihydropyridine receptor can bind to the ryanodine receptors in sarcoplasmic reticulum vesicles or in lipid bilayers and alter channel activity. Although micro-regions that participate in the functional interactions have been identified in each protein, the role of these regions and the molecular nature of the protein-protein interaction remain unknown. The trigger for Ca(2+) release through ryanodine receptors in cardiac muscle is a Ca(2+) influx through the L-type Ca(2+) channel. The Ca(2+) entering through the surface membrane Ca(2+) channels flows directly onto underlying ryanodine receptors and activates the channels. This was thought to be a relatively simple system compared with that in skeletal muscle. However, complexities are emerging and evidence has now been obtained for a bi-directional physical coupling between the proteins in cardiac as well as skeletal muscle. The molecular nature of this coupling remains to be elucidated.

Regulation of the calcium release channel from rabbit skeletal muscle by the nucleotides ATP, AMP, IMP and adenosine.

1. Nucleotide activation of skeletal muscle ryanodine receptors (RyRs) was studied in planar lipid bilayers in order to understand RyR regulation in vivo under normal and fatigued conditions. With 'resting' calcium (100 nM cytoplasmic and 1 mM luminal), RyRs had an open probability (P(o)) of approximately 0.01 in the absence of nucleotides and magnesium. ATP reversibly activated RyRs with P(o) at saturation (P(max)) approximately 0.33 and K(a) (concentration for half-maximal activation) approximately 0.36 mM and with a Hill coefficient (n(H)) of approximately 1.8 in RyRs when P(max) < 0.5 and approximately 4 when P(max) > 0.5. 2. AMP was a much weaker agonist (P(max) approximately 0.09) and adenosine was weaker still (P(max) approximately 0.01-0.02), whereas inosine monophosphate (IMP), the normal metabolic end product of ATP hydrolysis, produced no activation at all. 3. Adenosine acted as a competitive antagonist that reversibly inhibited ATP- and AMP-activated RyRs with n(H) approximately 1 and K(i) approximately 0.06 mM at [ATP] < 0.5 mM, increasing 4-fold for each 2-fold increase in [ATP] above 0.5 mM. This is explained by the binding of a single adenosine preventing the cooperative binding of two ATP or AMP molecules, with dissociation constants of 0.4, 0.45 and 0.06 mM for ATP, AMP and adenosine, respectively. Importantly, IMP (< or = 8 mM) had no inhibitory effect whatsoever on ATP-activated RyRs. 4. Mean open (tau(o)) and closed (tau(c)) dwell-times were more closely related to P(o) than to the nucleotide species or individual RyRs. At P(o) < 0.2, RyR regulation occurred via changes in tau(c), whereas at higher P(o) this also occurred via changes in tau(o). The detailed properties of activation and competitive inhibition indicated complex channel behaviour that could be explained in terms of a model involving interactions between different subunits of the RyR homotetramer. 5. The results also show how deleterious adenosine accumulation is to the function of RyRs in skeletal muscle and, by comparison with voltage sensor-controlled Ca(2+) release, indicate that voltage sensor activation requires ATP binding to the RyR to be effective.

Phosphate ion channels in sarcoplasmic reticulum of rabbit skeletal muscle.

1. Phosphate ions (P(i)) enter intracellular Ca2+ stores and precipitate Ca2+. Since transport pathways for P(i) across the membrane of intracellular calcium stores have not been identified and anion channels could provide such a pathway, we have examined the P(i) conductance of single anion channels from the sarcoplasmic reticulum (SR) of rabbit skeletal muscle using the lipid bilayer technique. 2. Two anion channels in skeletal muscle SR, the small conductance (SCl) and big conductance (BCl) chloride channels, were both found to have a P(i) conductance of 10 pS in 50 mM P(i). The SCl channel is a divalent anion channel which can pass HPO4(2-) as well as SO4(2-) (60 pS in 100 mM free SO4(2-)). The BCl channel is primarily a monovalent anion channel. The SCl and BCl channels are permeable to a number of small monovalent anions, showing minor selectivity between Cl-, I- and Br- (Cl- > I- > Br-) and relative impermeability to cations and large polyatomic anions (Cs+, Na+, choline+, Tris+, Hepes- and CH3O3S-). 3. The P(i) conductance of SCl and BCl channels suggests that both channel types could sustain the observed P(i) fluxes across the SR membrane. Comparison of the blocking effects of the phosphonocarboxylic acids, ATP and DIDS, on the anion channels with their effects on P(i) transport suggests that the SCl channel is the more likely candidate for the SR P(i) transport mechanism. 4. The SCl channel, with previously unknown function, provides a regulated pathway for P(i) across the SR membrane which would promote P(i) entry and thereby changes in the rapidly releasable Ca2+ store during onset and recovery from muscle fatigue. Anion channels may provide a pathway for P(i) movement into and out of Ca2+ stores in general.

Effects of cytoplasmic and luminal pH on Ca(2+) release channels from rabbit skeletal muscle.

Ryanodine receptor (RyR)-Ca(2+) release channels from rabbit skeletal muscle were incorporated into lipid bilayers. The effects of cytoplasmic and luminal pH were studied separately over the pH range 5-8, using half-unit intervals. RyR activity (at constant luminal pH of 7.5) was inhibited at acidic cytoplasmic pH, with a half-inhibitory pH (pH(I)) approximately 6.5, irrespective of bilayer potential and of whether the RyRs were activated by cytoplasmic Ca(2+) (50 microM), ATP (2 or 5 mM), or both. Inhibition occurred within approximately 1 s and could be fully reversed within approximately 1 s after brief inhibition or within approximately 30-60 s after longer exposure to acidic cytosolic pH. There was no evidence of any hysteresis in the cytoplasmic pH effect. Ryanodine-modified channels were less sensitive to pH inhibition, with pH(I) at approximately 5.5, but the inhibition was similarly reversible. Steady-state open and closed dwell times of RyRs during cytoplasmic pH inhibition suggest a mechanism where the binding of one proton inhibits the channel and the binding of two to three additional protons promotes further inhibited states. RyR activity was unaffected by luminal pH in the pH range 7.5 to 6.0. At lower luminal pH (5-5.5) most RyRs were completely inhibited, and raising the pH again produced partial to full recovery in only approximately 50% of cases, with the extent of recovery not detectably different between pH 7.5 and pH 9. The results indicate that isolated skeletal muscle RyRs are not inhibited as strongly by low cytoplasmic and luminal pH, as suggested by previous single-channel studies.

Activation and inhibition of skeletal RyR channels by a part of the skeletal DHPR II-III loop: effects of DHPR Ser687 and FKBP12.

Peptides, corresponding to sequences in the N-terminal region of the skeletal muscle dihydropyridine receptor (DHPR) II-III loop, have been tested on sarcoplasmic reticulum (SR) Ca2+ release and ryanodine receptor (RyR) activity. The peptides were: A1, Thr671-Leu690; A2, Thr671-Leu690 with Ser687 Ala substitution; NB, Gly689-Lys708 and A1S, scrambled A1 sequence. The relative rates of peptide-induced Ca2+ release from normal (FKBP12+) SR were A2 > A1 > A1S > NB. Removal of FKBP12 reduced the rate of A1-induced Ca2+ release by approximately 30%. A1 and A2 (but not NB or A1S), in the cytoplasmic (cis) solution, either activated or inhibited single FKBP12+ RyRs. Maximum activation was seen at -40 mV, with 10 microM A1 or 50 nM A2. The greatest A1-induced increase in mean current (sixfold) was seen with 100 nM cis Ca2+. Inhibition by A1 was greatest at +40 mV (or when permeant ions flowed from cytoplasm to SR lumen) with 100 microM cis Ca2+, where channel activity was almost fully inhibited. A1 did not activate FKBP12-stripped RyRs, although peptide-induced inhibition remained. The results show that peptide A activation of RyRs does not require DHPR Ser687, but required FKBP12 binding to RyRs. Peptide A must interact with different sites to activate or inhibit RyRs, because current direction-, voltage-, cis [Ca2+]-, and FKBP12-dependence of activation and inhibition differ.

ATP inhibition and rectification of a Ca2+-activated anion channel in sarcoplasmic reticulum of skeletal muscle.

We describe ATP-dependent inhibition of the 75-105-pS (in 250 mM Cl-) anion channel (SCl) from the sarcoplasmic reticulum (SR) of rabbit skeletal muscle. In addition to activation by Ca2+ and voltage, inhibition by ATP provides a further mechanism for regulating SCl channel activity in vivo. Inhibition by the nonhydrolyzable ATP analog 5'-adenylylimidodiphosphate (AMP-PNP) ruled out a phosphorylation mechanism. Cytoplasmic ATP (approximately 1 mM) inhibited only when Cl- flowed from cytoplasm to lumen, regardless of membrane voltage. Flux in the opposite direction was not inhibited by 9 mM ATP. Thus ATP causes true, current rectification in SCl channels. Inhibition by cytoplasmic ATP was also voltage dependent, having a K(I) of 0.4-1 mM at -40 mV (Hill coefficient approximately 2), which increased at more negative potentials. Luminal ATP inhibited with a K(I) of approximately 2 mM at +40 mV, and showed no block at negative voltages. Hidden Markov model analysis revealed that ATP inhibition 1) reduced mean open times without altering the maximum channel amplitude, 2) was mediated by a novel, single, voltage-independent closed state (approximately 1 ms), and 3) was much less potent on lower conductance substates than the higher conductance states. Therefore, the SCl channel is unlikely to pass Cl- from cytoplasm to SR lumen in vivo, and balance electrogenic Ca2+ uptake as previously suggested. Possible roles for the SCl channel in the transport of other anions are discussed.

Inactivation of Ca2+ release channels (ryanodine receptors RyR1 and RyR2) with rapid steps in [Ca2+] and voltage.

The transient responses of sheep cardiac and rabbit skeletal ryanodine receptors (RyRs) to step changes in membrane potential and cytosolic [Ca2+] were measured. Both cardiac and skeletal RyRs have two voltage-dependent inactivation processes (tau approximately 1-3 s at +40 mV) that operate at opposite voltage extremes. Approximately one-half to two-thirds of RyRs inactivated when the bilayer voltage was stepped either way between positive and negative values. Inactivation was not detected (within 30 s) in RyRs with Po less than 0.2. Inactivation rates increased with intraburst open probability (Po) and in proportion to the probability of a long-lived, RyR open state (P(OL)) RyR inactivation depended on P(OL) and not on the particular activator (Ca2+ (microM), ATP, caffeine, and ryanodine), inhibitor (mM Ca2+ and Mg2+), or gating mode. The activity of one-half to two-thirds of RyRs declined (i.e., the RyRs inactivated) after [Ca2+] steps from subactivating (0.1 microM) to activating (1-100 microM) levels. This was due to the same inactivation mechanism responsible for inactivation after voltage steps. Both forms of inactivation had the same kinetics and similar dependencies on Po and voltage. Moreover, RyRs that failed to inactivate after voltage steps also did not inactivate after [Ca2+] steps. The inactivating response to [Ca2+] steps (0.1-1 microM) was not RyRs "adapting" to steady [Ca2+] after the step, because a subsequent step from 1 to 100 microM failed to reactivate RyRs.

Reduced inhibitory effect of Mg2+ on ryanodine receptor-Ca2+ release channels in malignant hyperthermia.

Malignant hyperthermia (MH) is a potentially fatal, inherited skeletal muscle disorder in humans and pigs that is caused by abnormal regulation of Ca2+ release from the sarcoplasmic reticulum (SR). MH in pigs is associated with a single mutation (Arg615Cys) in the SR ryanodine receptor (RyR) Ca2+ release channel. The way in which this mutation leads to excessive Ca2+ release is not known and is examined here. Single RyR channels from normal and MH-susceptible (MHS) pigs were examined in artificial lipid bilayers. High cytoplasmic (cis) concentrations of either Ca2+ or Mg2+ (>100 microM) inhibited channel opening less in MHS RyRs than in normal RyRs. This difference was more prominent at lower ionic strength (100 mM versus 250 mM). In 100 mM cis Cs+, half-maximum inhibition of activity occurred at approximately 100 microM Mg2+ in normal RyRs and at approximately 300 microM Mg2+ in MHS RyRs, with an average Hill coefficient of approximately 2 in both cases. The level of Mg2+ inhibition was not appreciably different in the presence of either 1 or 50 microM activating Ca2+, showing that it was not substantially influenced by competition between Mg2+ and Ca2+ for the Ca2+ activation site. Even though the absolute inhibitory levels varied widely between channels and conditions, the inhibitory effects of Ca2+ and Mg2+ were virtually identical for the same conditions in any given channel, indicating that the two cations act at the same low-affinity inhibitory site. It seems likely that at the cytoplasmic [Mg2+] in vivo (approximately 1 mM), this Ca2+/Mg2+-inhibitory site will be close to fully saturated with Mg2+ in normal RyRs, but less fully saturated in MHS RyRs. Therefore MHS RyRs should be more sensitive to any activating stimulus, which would readily account for the development of an MH episode.

Magnesium inhibition of ryanodine-receptor calcium channels: evidence for two independent mechanisms.

The gating of ryanodine receptor calcium release channels (RyRs) depends on myoplasmic Ca2+ and Mg2+ concentrations. RyRs from skeletal and cardiac muscle are activated by microm Ca2+ and inhibited by mm Ca2+ and Mg2+. 45Ca2+ release from skeletal SR vesicles suggests two mechanisms for Mg2+-inhibition (Meissner, Darling & Eveleth, 1986, Biochemistry 25:236-244). The present study investigates the nature of these mechanisms using measurements of single-channel activity from cardiac- and skeletal RyRs incorporated into planar lipid bilayers. Our measurements of Mg2+- and Ca2+-dependent gating kinetics confirm that there are two mechanisms for Mg2+ inhibition (Type I and II inhibition) in skeletal and cardiac RyRs. The mechanisms operate concurrently, are independent and are associated with different parts of the channel protein. Mg2+ reduces Po by competing with Ca2+ for the activation site (Type-I) or binding to more than one, and probably two low affinity inhibition sites which do not discriminate between Ca2+ and Mg2+ (Type-II). The relative contributions of the two inhibition mechanisms to the total Mg2+ effect depend on cytoplasmic [Ca2+] in such a way that Mg2+ inhibition has the properties of Types-I and II inhibition at low and high [Ca2+] respectively. Both mechanisms are equally important when [Ca2+] = 10 microm in cardiac RyRs or 1 microm in skeletal RyRs. We show that Type-I inhibition is not the sole mechanism responsible for Mg2+ inhibition, as is often assumed, and we discuss the physiological implications of this finding.