Activation of cardiac ryanodine receptors by cardiac glycosides

This study investigated the effects of cardiac glycosides on single-channel activity of the cardiac sarcoplasmic reticulum (SR) Ca2+ release channels or ryanodine receptor (RyR2) channels and how this action might contribute to their inotropic and/or toxic actions. Heavy SR vesicles isolated from canine left ventricle were fused with artificial planar lipid bilayers to measure single RyR2 channel activity. Digoxin and actodigin increased single-channel activity at low concentrations normally associated with therapeutic plasma levels, yielding a 50% of maximal effect of approximately 0.2 nM for each agent. Channel activation by glycosides did not require MgATP and occurred only when digoxin was applied to the cytoplasmic side of the channel. Similar results were obtained in human RyR2 channels; however, neither the crude skeletal nor the purified cardiac channel was activated by glycosides. Channel activation was dependent on [Ca2+] on the luminal side of the bilayer with maximal stimulation occurring between 0.3 and 10 mM. Rat RyR2 channels were activated by digoxin only at 1 microM, consistent with the lower sensitivity to glycosides in rat heart. These results suggest a model in which RyR2 channel activation by digoxin occurs only when luminal [Ca2+] was increased above 300 microM (in the physiological range). Consequently, increasing SR load (by Na+ pump inhibition) serves to amplify SR release by promoting direct RyR2 channel activation via a luminal Ca2+-sensitive mechanism. This high-affinity effect of glycosides could contribute to increased SR Ca2+ release and might play a role in the inotropic and/or toxic actions of glycosides in vivo.     

Redox-sensitive stimulation of type-1 ryanodine receptors by the scorpion toxin maurocalcine

The scorpion toxin maurocalcine acts as a high affinity agonist of the type-1 ryanodine receptor expressed in skeletal muscle. Here, we investigated the effects of the reducing agent dithiothreitol or the oxidizing reagent thimerosal on type-1 ryanodine receptor stimulation by maurocalcine. Maurocalcine addition to sarcoplasmic reticulum vesicles actively loaded with calcium elicited Ca²⁺ release from native vesicles and from vesicles pre-incubated with dithiothreitol; thimerosal addition to native vesicles after Ca²⁺ uptake completion prevented this response. Maurocalcine enhanced equilibrium [³H]-ryanodine binding to native and to dithiothreitol-treated reticulum vesicles, and increased 5-fold the apparent K_i for Mg²⁺ inhibition of [³H]-ryanodine binding to native vesicles. Single calcium release channels incorporated in planar lipid bilayers displayed a long-lived open sub-conductance state after maurocalcine addition. The fractional time spent in this sub-conductance state decreased when lowering cytoplasmic [Ca²⁺] from 10 μM to 0.1 μM or at cytoplasmic [Mg²⁺] ≥ 30 μM. At 0.1 μM [Ca²⁺], only channels that displayed poor activation by Ca²⁺ were readily activated by 5 nM maurocalcine; subsequent incubation with thimerosal abolished the sub-conductance state induced by maurocalcine. We interpret these results as an indication that maurocalcine acts as a more effective type-1 ryanodine receptor channel agonist under reducing conditions.     

Activation of purified cardiac ryanodine receptors by dihydropyridine agonists

Prior observations have raised the possibility that dihydropyridine (DHP) agonists directly affect the sarcoplasmic reticulum (SR) cardiac Ca(2+) release channel [i.e., ryanodine receptor (RyR)]. In single-channel recordings of purified canine cardiac RyR, both DHP agonists (-)-BAY K 8644 and (+)-SDZ202-791 increased the open probability of the RyR when added to the cytoplasmic face of the channel. Importantly, the DHP antagonists nifedipine and (-)-SDZ202-791 had no competitive blocking effects either alone or after channel activation with agonist. Thus there is a stereospecific effect of SDZ202-791, such that the agonist activates the channel, whereas the antagonist has little effect on channel activity. Further experiments showed that DHP agonists changed RyR activation by suppressing Ca(2+)-induced inactivation of the channel. We concluded that DHP agonists can also influence RyR single-channel activity directly at a unique allosteric site located on the cytoplasmic face of the channel. Similar results were obtained in human purified cardiac RyR. An implication of these data is that RyR activation by DHP agonists is likely to cause a loss of Ca(2+) from the SR and to contribute to the negative inotropic effects of these agents reported by other investigators. Our results support this notion that the negative inotropic effects of DHP agonists result in part from direct alteration in the activity of RyRs.     

Activation of the ryanodine receptor Ca2+ release channel of sarcoplasmic reticulum by a novel scorpion venom.

We identified a peptide fraction from the venom of the scorpion Buthotus hottentota that stimulated binding of [3H]ryanodine to ryanodine receptors of skeletal and cardiac sarcoplasmic reticulum and brain microsomes in a highly specific manner. Activity was concentrated in a peptide fraction of Mr 5,000-8,000. Assuming a single active peptide in this fraction, we estimated a dissociation constant of 20-30 nM for the interaction of the peptide with the ryanodine receptor. The whole venom and the purified fraction activated skeletal ryanodine receptor Ca2+ release channels incorporated into planar lipid bilayers. The venom produced a 10-fold increase in the mean open time and induced the appearance of a long lasting subconductance state not seen in controls. Changes were reversible and could be induced by the partially purified venom fraction. This novel scorpion venom should be helpful in establishing the role of ryanodine receptors in the initiation of intracellular Ca2+ release in striated muscle and in nonmuscle cells containing functional ryanodine receptors such as neurons and secretory cells.     

Simultaneous modifications of sodium channel gating by two scorpion toxins.

The effects of purified scorpion toxins from two different species on the kinetics of sodium currents were evaluated in amphibian myelinated nerves under voltage clamp. A toxin from Leiurus quinquestriatus slowed and prevented sodium channel inactivation, exclusively, and a toxin from Centruroides sculpturatus Ewing reduced transient sodium currents during a maintained depolarization, and induced a novel inward current that appeared following repolarization, as previously reported by Cahalan (1975, J. Physiol. [Lond.]. 244:511-534) for the crude scorpion venom. Both of these effects were observed in fibers treated with both of these toxins, and the kinetics of the induced current were modified in a way that showed that the same sodium channels were modified simultaneously by both toxins. Although the toxins can act on different sites, the time course of the action of C. sculpturatus toxin was accelerated in the presence of the L. quinquestriatus toxin, indicating some form of interaction between the two toxin binding sites.     

Structural and functional differences of two toxins from the scorpion Pandinus imperator

The Pandinotoxins, PiTX-K alpha and PiTX-K beta, are members of the Charybdotoxin family of scorpion toxins that can be used to characterize K+ channels. PiTX-K alpha differs from PiTX-K beta, another peptide from Pandinus imperator, by one residue (P10E). When the two toxins are compared in a physiological assay, the affinity of PiTX-K beta for voltage-gated, rapidly inactivating K+ channels in dorsal root ganglia (DRG) neurons is 800-fold lower than that of PiTX-K alpha (K alpha-IC50 = 8.0 nM versus K beta-IC50 = 6,500 nM). To understand this difference, the three-dimensional structure of PiTX-K beta was determined by nuclear magnetic resonance (NMR) spectroscopy and compared to that of PiTX-K alpha. This comparison shows that structural differences between the two toxins occur at a residue that is critical for blocking K+ channels (K27) as well as at the site of the natural mutation (P10E). In PiTX-K beta, the negatively charged carboxylate oxygen of E10 can approach the positive charge of K27 and presumably reduces the net positive charge in this region of the toxin. This is likely the reason why PiTX-K beta binds K+ channels from DRG neurons with a much lower affinity than does PiTX-K alpha.     

Characterization of scorpion alpha-like toxin group using two new toxins from the scorpion Leiurus quinquestriatus hebraeus.

Two novel toxins, Lqh6 and Lqh7, isolated from the venom of the scorpion Leiurus quinquestriatus hebraeus, have in their sequence a molecular signature (8Q/KPE10) associated with a recently defined group of alpha-toxins that target Na channels, namely the alpha-like toxins [reviewed in Gordon, D., Savarin, P., Gurevitz, M. & Zinn-Justin, S. (1998) J. Toxicol. Toxin Rev. 17, 131-159]. Lqh6 and Lqh7 are highly toxic to insects and mice, and inhibit the binding of alpha-toxins to cockroach neuronal membranes. Although they kill rodents by intracerebroventricular injection, they do not inhibit the binding of antimammal alpha-toxins (e.g. Lqh2) to rat brain synaptosomes, not even at high concentrations. Furthermore, in voltage-clamp experiments, rat brain Na channels IIA (rNav1.2A) expressed in Xenopus oocytes are not affected by Lqh6 nor by Lqh7 below 3 micro m. In contrast, muscular Na channels (rNav1.4 and hNav1.5) expressed in the same cells respond to nanomolar concentrations of Lqh6 and Lqh7 by slowing of Na current inactivation and a leftward shift of the peak conductance-voltage curve. The structural and pharmacological properties of the new toxins are compared to those of other scorpion alpha-toxins in order to re-examine the hallmarks previously set for the alpha-like toxin group.     

Activation of ryanodine receptors by flash photolysis of caged Ca2+.

Flash photolysis of DM-nitrophen generates an extremely large [Ca2+] transient ("Ca2+ spike") at the start of each Ca2+ "step." The Ca2+ spike greatly increases the speed of activation of the ryanodine receptor channel ("supercharging") and could be responsible for apparent channel adaptation.     

Activation of cardiac ryanodine receptors by the calcium channel agonist FPL-64176

We investigated the possibility that the Ca(2+) channel agonist FPL-64176 (FPL) might also activate the cardiac sarcoplasmic reticulum (SR) Ca(2+) release channel ryanodine receptor (RyR). The effects of FPL were tested on single channel activity of purified and crude vesicular RyR (RyR2) isolated from human and dog hearts using the planar lipid bilayer technique. FPL (100-200 microM) increased single channel open probability (P(o)) when added to the cytoplasmic side of the channel (P(o) = 0.070 +/- 0.021 in control RyR2; 0.378 +/- 0.086 in 150 microM FPL, n = 9, P < 0.01) by prolonging open times and decreasing closed times without changing current magnitude. FPL had no effect on P(o) when added to the trans (luminal) side of the bilayer (P(o) = 0.079 +/- 0.036 in control and 0.103 +/- 0.066 in FPL, n = 4, no significant difference). The bell-shaped [Ca(2+)] dependence of [(3)H]ryanodine binding and of P(o) was altered by FPL, suggesting that the mechanism by which FPL increases channel activity is by an increase in Ca(2+)-induced activation at low [Ca(2+)] (without a change in threshold) and suppression of Ca(2+)-induced inactivation at high [Ca(2+)]. However, the fact that inactivation was restored at elevated [Ca(2+)] suggests a competitive interaction between Ca(2+) and FPL on inactivation. FPL had no effect on RyR skeletal channels (RyR1), where P(o) was 0.039 +/- 0.005 in control versus 0.030 +/- 0.006 in 150 microM FPL (no significant difference). These results suggest that, in addition to its ability to activate the L-type Ca(2+) channels, FPL activates cardiac RyR2 primarily by reducing the Ca(2+) sensitivity of inactivation.     

Oxidation and reduction of pig skeletal muscle ryanodine receptors

Time-dependent effects of cysteine modification were compared in skeletal ryanodine receptors (RyRs) from normal pigs and RyR(MH) (Arg(615) to Cys(615)) from pigs susceptible to malignant hyperthermia, using the oxidizing reagents 4,4'-dithiodipyridine (4, 4'-DTDP) and 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) or the reducing agent dithiothreitol (DTT). Normal and RyR(MH) channels responded similarly to all reagents. DTNB (1 mM), either cytoplasmic (cis) or luminal (trans), or 1 mM 4,4'-DTDP (cis) activated RyRs, introducing an additional long open time constant. 4,4'-DTDP (cis), but not DTNB, inhibited channels after >5 min. Activation and inhibition were relieved by DTT (1-10 mM). DTT (10 mM, cytoplasmic or luminal), without oxidants, activated RyRs, and activation reversed with 1 mM DTNB. Control RyR activity was maintained with 1 mM DTNB and 10 mM DTT present on the same or opposite sides of the bilayer. We suggest that 1) 4,4'-DTDP and DTNB covalently modify RyRs by oxidizing activating or inhibiting thiol groups; 2) a modified thiol depresses mammalian skeletal RyR activity under control conditions; 3) both the activating thiols and the modified thiols, accessible from either cytoplasm or lumen, reside in the transmembrane region; 4) some cardiac sulfhydryls are unavailable in skeletal RyRs; and 5) Cys(615) in RyR(MH) is functionally unimportant in redox cycling.     

Isolation and characterization of two toxins from the Mexican scorpion Centruroides limpidus limpidus Karsch

1. Several toxic polypeptides were found in the venom of the scorpion Centruroides limpidus limpidus. Comparative studies of the potency of the venom in different strains of mice were conducted. 2. A new type of toxin (component II.9), specific for crustaceans (crayfish and isopods), was isolated from this scorpion and was shown to have the following N-terminal amino acid sequence: Lys-Lys-Asp-Gly-Tyr-Leu-Val-Asn-Lys-Tyr-Thr-Gly-Cys-Lys-Val-Asn-Cys- Tyr-Lys-Leu-Gly-Glu-Asn-Lys-Phe-Cys-Asn-Arg-Glu-. 3. A polypeptide toxic to mice (component II.6) from this venom was shown to have the following N-terminal sequence: Lys-Glu-Gly-Tyr-Leu-Val-Asn-His-Ser-Thr-Gly-Cys-Lys-Tyr- Glu-Cys-Tyr-Lys-Leu-Gly-Asp-Asn-Asp-Tyr-Cys-Leu-Arg-Glu-Cys-Lys-. 4. In cultured chick dorsal root ganglion cells, 1 microM of toxin II.6 was shown to reduce the size of sodium currents and to slow-down their activation-inactivation kinetics. The toxin had also a depressive action on the classical Ca2+ current activated at high membrane potentials (greater than 0 mV).     

Nitroxyl triggers Ca2+ release from skeletal and cardiac sarcoplasmic reticulum by oxidizing ryanodine receptors

The biological activity of nitric oxide (NO) and NO-donors has been extensively investigated yet few studies have examined those of nitroxyl (HNO) species even though both exist in chemical equilibrium but oxidize thiols by different reaction mechanisms: S-nitrosation versus disulfide bond formation. Here, sodium trioxodinitrate (Na2N2O3; Angeli's salt; ANGS) was used as an HNO donor to investigate its effects on skeletal (RyR1) and cardiac (RyR2) ryanodine receptors. At steady-state concentrations of nanomoles/L, HNO induced a rapid Ca2+ release from sarcoplasmic reticulum (SR) vesicles then the reducing agent dithiothreitol (DTT) reversed the oxidation by HNO resulting in Ca2+ re-uptake by SR vesicles. With RyR1 channel proteins reconstituted in planar bilayers, HNO added to the cis-side increased the open probability (Po) from 0.056+/-0.026 to 0.270+/-0.102 (P<0.005, n=4) then DTT (3 mM) reduced Po to 0.096+/-0.040 (P<0.01, n=4). In parallel experiments, the time course of HNO production from ANGS was monitored by EPR and UV spectroscopy and compared with the rate of SR Ca2+ release indicating that picomolar concentrations of HNO triggered SR Ca2+ release. Controls showed that the hydroxyl radical scavenger, phenol did not alter ANGS-induced SR Ca2+ release, indicating that hydroxyl radical production from ANGS did not account for Ca2+ release from the SR. The findings indicate that HNO is a more potent activator of RyR1 than NO and that HNO activation of RyRs may contribute to NO's activation of RyRs and to the therapeutic effects of HNO-releasing prodrugs in heart failure.     

Activation of skeletal muscle nicotinic acetylcholine receptors

Summary and Conclusions Work over the past ten years has greatly increased our understanding of both the structure and function of the muscle nicotinic acetylcholine receptor. There is a strongly supported general picture of how the receptor functions: agonist binds rapidly to sites of low affinity and channel opening occurs at a rate comparable to the agonist dissociation rate. Channel closing is slow, so the channel has a high probability of being open if both agonist-binding sites are occupied by ACh. Results of expression studies have shown that each subunit can influence AChR activation and have given a structural basis for the major physiological change known for muscle AChR, the developmental change in AChR activation. These general statements notwithstanding, there are still major areas of uncertainty which limit our understanding. We have emphasized these areas of uncertainty in this review, to indicate what needs to be done.First, the quantitative estimates of rate constants are not as strongly supported as they should be. The major reasons are twofold—uncertainties about the interpretation of components in the kinetic data and difficulties of resolving brief events. As a result, any inferences about the functional consequences of structural alterations must remain tenuous.Second, the functional behavior of individual AChRs is not as well understood as it should be. The kinetic behavior of an individual receptor clearly can be complex (section II). In addition, there is evidence that superimposed on this complexity there may be stable and kinetically distinguishable populations of receptors (section III). Until the basis for the kinetically defined populations is clarified, kinetic parameters for receptors of defined structure cannot be unambiguously obtained.Finally, it is not surprising that the studies of AChR of altered structure have not given definitive results. Two reasons should be apparent from the preceding points: there is not a fully supported approach for kinetic analysis, and the normal population may not be clearly defined. An additional complication is also emerging, in that the available data support the idea that specific residues distributed over all subunits may influence AChR activation. This possibility renders the task of analysis that much more difficult.The muscle nicotinic AChR has served as a prototype for the family of transmitter-gated membrane channels, which includes the muscle and neuronal nicotinic receptors, the GABA_A, the glycine and possibly the non-NMDA excitatory amino acid receptor (Stroud et al., 1990). It is interesting to note that the functional properties of the GABA_A receptor, probably the best-studied of the other members of the family are rather similar. In particular, opentime and burst durations show multiple components interpreted as reflecting openings of singly and doubly liganded receptors (Mathers & Wang, 1988; Macdonald et al., 1989), the distribution of gaps indicates a relatively complex gating scheme (Twyman et al., 1990; Weiss & Magleby, 1989), and multiple kinetic modes are likely to exist (Newland et al., 1991). The situation with regards to the effects of GABA_A receptor subunit stoichiometry is more complex than for muscle AChR (e.g., Luddens & Wisden, 1991), perhaps similar to that found for neuronal nicotinic AChR (Papke et al., 1989; Luetje et al., 1990; Luetje & Patrick, 1991). Overall, it appears that the unresolved questions about the muscle nicotinic AChR are not indications that this is an exceptionally complicated transmitter-gated channel. Rather, it appears to be a relatively straightforward member of the family, and the lessons we learn from studying it are likely to be directly applicable to other receptors.We thank many friends for discussion, including Tony Auerbach, Paul Brehm, Jim Dilger, Meyer Jackson, and Chuck Stevens who told us about data before publication. Research in the authors' laboratories is supported by grants from the NIH (CL and JHS) and the AHA (CL).     

Ca2+对骨骼肌钙释放通道的调节

钙释放通道（calcium release channel）又称Ryanodine受体（RyR）,是细胞内质网膜上介导细胞内钙信号转导的离子通道。RyR1在骨骼肌细胞的兴奋-收缩偶联过程中起重要作用,是肌质网快速释放Ca^2＋的通道。许多调节因素,如一些内源性蛋白（FK结合蛋白、钙调素、钙结合蛋白）和一些离子（Ca^2＋、Mg^2＋）,通过不同的作用位点与RyR1结合,调控RyR1的结构与功能。研究表明,Ca^2＋是众多调节RyR1因素中的核心成分和前提条件,其对RyR1的结构与功能有重要的调控作用。     

cDNA cloning, sequence analysis and molecular modeling of a new peptide from the scorpion Buthotus saulcyi venom

In this study, the cDNA of a new peptide from the venom of the scorpion, Buthotus saulcyi, was cloned and sequenced. It codes for a 64 residues peptide (Bsaul1) which shares high sequence similarity with depressant insect toxins of scorpions. The differences between them mainly appear in the loop1 which connects the beta-strand1 to the alpha-helix and seems to be functionally important in long chain scorpion neurotoxins. This loop is three amino acids longer in Bsaul1 compared to other depressant toxins. A comparative amino acid sequence analysis done on Bsaul1 and some of alpha-, beta-, excitatory and depressant toxins of scorpions showed that Bsaul1 contains all the residues which are highly conserved among long chain scorpion neurotoxins. Structural model of Bsaul1 was generated using Ts1 (a beta-toxin that competes with the depressant insect toxins for binding to Na(+) channels) as template. According to the molecular model of Bsaul1, the folding of the polypeptide chain is being composed of an anti-parallel three-stranded beta-sheet and a stretch of alpha- helix, tightly bound by a set of four disulfide bridges. A striking similarity in the spatial arrangement of some critical residues was shown by superposition of the backbone conformation of Bsaul1 and Ts1.     

Rapamycin inhibits activation of ryanodine receptors from skeletal muscle by the fatty acyl CoA-acyl CoA binding protein complex.

We previously showed (Fulceri et al., Biochem. J. 325, 423, 1997) that the fatty acyl CoA ester palmitoyl CoA (PCoA) complexed with a molar excess of its cytosolic binding protein (ACBP) causes a discrete Ca(2+) efflux or allows Ca(2+) release by suboptimal caffeine concentrations, in the Ca(2+)-preloaded terminal cisternae fraction (TC) from rabbit skeletal muscle, by activating ryanodine receptor Ca(2+) release channels (RyRC). We show here that both effects were abolished by pretreating TC with the FKBP12 ligand rapamycin (20 microM). Moreover, rapamycin reversed the Ca(2+) release induced by combined treatment with 3 mM caffeine and the PCoA-ACBP complex. Rapamycin also reduced the Ca(2+)-releasing activity by PCoA alone. Under the above experimental conditions, rapamycin removed FKBP12 from the TC membranes, as revealed by Western blot analysis. We conclude that FKBP12 associated with RyRC in the TC membrane participates in the activation of the Ca(2+) channel by fatty acyl CoA esters.     

Comparative pharmacology and cloning of two novel arachnid sodium channels: Exploring the adaptive insensitivity of scorpion to its toxins

Scorpion toxins have been found lacking effect on Na(+) current of its own sodium channel, whereas the molecular mechanism remains mystery. In this study, the binding affinity of pharmacologically distinct scorpion toxins was found much weaker to scorpion (Buthus martensii) nerve synaptosomes than to spider (Ornithoctonus huwena) ones. The sodium channel cDNA from these two species were further cloned. The deduced proteins contain 1871 and 1987 amino acids respectively. Several key amino acid substitutions, i.e., A1610V, I1611L and S1617K, are found in IVS3-S4 constituting receptor site-3, and for receptor site-4, two residues (Leu-Pro) are inserted near IIS4 of scorpion sodium channel.