Jonathan J. Abramson

Hydrogen Peroxide Stimulates the Ca2+ Release Channel from Skeletal Muscle Sarcoplasmic Reticulum

Hydrogen peroxide (H2O2) at millimolar concentrations induces Ca2+ release from actively loaded sarcoplasmic reticulum vesicles and induces biphasic [3H]ryanodine binding behavior. High affinity [3H]ryanodine binding is enhanced at concentrations from 100 μM to 10 mM (3-4-fold). At H2O2 concentrations greater than 10 mM, equilibrium binding is inhibited. H2O2 decreased the kd for [3H]ryanodine binding by increasing its association rate, while having no effect on the rate of dissociation of [3H]ryanodine from its receptor. H2O2 (1 mM) also reduced the EC50 for Ca2+ activation from 632 nM to 335 nM. These effects were completely abolished in the presence of catalase, ruthenium red, and/or Mg2+ (mM). H2O2-stimulated [3H]ryanodine binding is not further enhanced by either doxorubicin or caffeine. The direct interaction between H2O2 and the Ca2+ release mechanism was further demonstrated in single-channel reconstitution experiments. Peroxide, at submillimolar concentrations, activated the Ca2+ release channel following fusion of a sarcoplasmic reticulum vesicle to a bilayer lipid membrane. At millimolar concentrations of peroxide, Ca2+ channel activity was inhibited. Peroxide stimulation of Ca2+ channel activity was reversed by the thiol reducing agent dithiothreitol. Paralleling peroxide induced activation of ryanodine binding, Ca2+ transport, and single Ca2+ channel activity, it was observed that the ryanodine receptor formed large disulfidelinked protein complexes that dissociated upon addition of dithiothreitol.

Critical sulfhydryls regulate calcium release from sarcoplasmic reticulum

Rapid Ca2+ release from the sarcoplasmic reticulum (SR) can be triggered by either binding of heavy metals to a sulfhydryl (SH) group or by catalyzing the oxidation of endogenous groups to a disulfide. Ca2+ release has been monitored directly using isolated vesicle preparations or indirectly by monitoring phasic contractions in a skinned fiber preparation. SH oxidation triggered by addition of Cu2+ /mercaptans, phthalocyanine dyes, reactive disulfides, and various anthraquinones appears to involve a direct interaction with the Ca2+ release protein from the SR. A model is presented in which reversible oxidation and reduction of endogenous SH groups results in the opening and closing of the Ca2+ release channel from the SR.

Selenoprotein N is required for ryanodine receptor calcium release channel activity in human and zebrafish muscle

Mutations affecting the seemingly unrelated gene products, SepN1, a selenoprotein of unknown function, and RyR1, the major component of the ryanodine receptor intracellular calcium release channel, result in an overlapping spectrum of congenital myopathies. To identify the immediate developmental and molecular roles of SepN and RyR in vivo, loss-of-function effects were analyzed in the zebrafish embryo. These studies demonstrate the two proteins are required for the same cellular differentiation events and are needed for normal calcium fluxes in the embryo. SepN is physically associated with RyRs and functions as a modifier of the RyR channel. In the absence of SepN, ryanodine receptors from zebrafish embryos or human diseased muscle have altered biochemical properties and have lost their normal sensitivity to redox conditions, which likely accounts for why mutations affecting either factor lead to similar diseases.

Glutathione Modulates Ryanodine Receptor from Skeletal Muscle Sarcoplasmic Reticulum EVIDENCE FOR REDOX REGULATION OF THE Ca2+ RELEASE MECHANISM

In this report, we demonstrate the ability of the cellular thiol glutathione to modulate the ryanodine receptor from skeletal muscle sarcoplasmic reticulum. Reduced glutathione (GSH) inhibited Ca2+-stimulated [3H]ryanodine binding to the sarcoplasmic reticulum and inhibited the single-channel gating activity of the reconstituted Ca2+ release channel. The effects of GSH on both the [3H]ryanodine binding and single-channel measurements were dose-dependent, exhibiting an IC50 of ∼2.4 mM in binding experiments. Scatchard analysis demonstrated that GSH decreased the binding affinity of ryanodine for its receptor (increased Kd) and lowered the maximal binding occupancy (Bmax). In addition, GSH did not modify the Ca2+ dependence of [3H]ryanodine binding. In single-channel experiments, GSH (5-10 mM), added to the cis side of the bilayer lipid membrane, lowered the open probability (Po) of a Ca2+ (50 μM)-stimulated Ca2+ channel without modifying the single-channel conductance. Subsequent perfusion of the cis chamber with an identical buffer, containing 50 μM Ca2+ without GSH, re-established Ca2+-stimulated channel gating. GSH did not inhibit channel activity when added to the trans side of the bilayer lipid membrane. Similar to GSH, the thiol-reducing agents dithiothreitol and β-mercaptoethanol also inhibited high affinity [3H]ryanodine binding to sarcoplasmic reticulum membranes. In contrast to GSH, glutathione disulfide (GSSG) was a potent stimulator of high affinity [3H]ryanodine binding and it also stimulated the activity of the reconstituted single Ca2+ release channel. These results provide direct evidence that glutathione interacts with reactive thiols associated with the Ca2+ release channel/ryanodine receptor complex, which are located on the cytoplasmic face of the SR, and support previous observations (Liu, G, Abramson, J. J., Zable, A. C., and Pessah, I. N. (1994) Mol. Pharmacol. 45, 189-200) that reactive thiols may be involved in the gating of the Ca2+ release channel.

Skeletal muscle ryanodine receptor is a redox sensor with a well defined redox potential that is sensitive to channel modulators.

Hyperreactive sulfhydryl groups associated with the Ca2+ release protein from sarcoplasmic reticulum are shown to have a well defined reduction potential that is sensitive to the cellular environment. Ca2+ channel activators lower the redox potential of the ryanodine receptor, which favors the oxidation of thiols and the opening of the Ca2+ release protein. In contrast, channel inhibitors increase the redox potential, which favors the reduction of disulfides and the closure of the release protein. Modulation of redox potential of reactive thiols may be a general control mechanism by which sarcoplasmic/endoplasmic reticulum, ryanodine receptors/IP3 receptors, control cytoplasmic Ca2+ concentrations.

Oxidation induced by phthalocyanine dyes causes rapid calcium release from sarcoplasmic reticulum vesicles

The copper containing phthalocyanine dyes, alcian blue, copper phthalocyanine tetrasulfonic acid, and Luxol fast blue MBSN are found to induce rapid calcium efflux from actively loaded sarcoplasmic reticulum (SR) vesicles. Alcian blue (5 microM), with 1 mM free Mg2+ triggered Ca2+ efflux at rates greater than 20 nmol/mg of SR/s. As in the case of Ca2+ efflux induced by calcium, heavy metals, or SH oxidation with Cu2+/cysteine, efflux induced by phthalocyanines is also stimulated by adenine containing nucleotides and inhibited by millimolar Mg2+ and submicromolar ruthenium red (RR). In addition, analogs of RR, such as hexamminecobalt(III) chloride or hexammineruthenium(III) chloride also inhibit Ca2+ efflux but are effective at somewhat higher concentrations (approximately 50 microM). Calcium release stimulated by phthalocyanines is specific for SR derived from the terminal cisternae region rather than longitudinal SR. Preincubation of alcian blue with the reducing agents, sodium dithionite, dithiothreitol, or cysteine causes complete loss of Ca2+ release activity from SR vesicles. Reoxidation of the alcian blue leads to return of the Ca2+ release activity of the phthalocyanine dye. The copper containing phthalocyanine dyes appear to cause rapid Ca2+ release from SR vesicles by oxidizing sulfhydryl groups associated with the calcium release channel. Moreover, phthalocyanines appear to act by oxidizing a pair of neighboring sulfhydryls to a disulfide because subsequent additions of the reducing agent dithiothreitol promote the closure of the Ca2+ channel and calcium re-uptake.

Ranolazine stabilizes cardiac ryanodine receptors: A novel mechanism for the suppression of early afterdepolarization and torsades de pointes in long QT type 2

BACKGROUND Ranolazine (Ran) is known to inhibit multiple targets, including the late Na(+)current, the rapid delayed rectifying K(+)current, the L-type Ca(2+)current, and fatty acid metabolism. Functionally, Ran suppresses early afterdepolarization (EADs) and torsades de pointes (TdP) in drug-induced long QT type 2 (LQT2) presumably by decreasing intracellular [Na(+)](i) and Ca(2+)overload. However, simulations of EADs in LQT2 failed to predict their suppression by Ran. OBJECTIVE To elucidate the mechanism(s) whereby Ran alters cardiac action potentials (APs) and cytosolic Ca(2+)transients and suppresses EADs and TdP in LQT2. METHODS The known effects of Ran were included in simulations (Shannon and Mahajan models) of rabbit ventricular APs and Ca(2+)transients in control and LQT2 models and compared with experimental optical mapping data from Langendorff rabbit hearts treated with E4031 (0.5 μM) to block the rapid delayed rectifying K(+)current. Direct effects of Ran on cardiac ryanodine receptors (RyR2) were investigated in single channels and changes in Ca(2+)-dependent high-affinity ryanodine binding. RESULTS Ran (10 μM) alone prolonged action potential durations (206 ± 4.6 to 240 ± 7.8 ms; P <0.05); E4031 prolonged action potential durations (204 ± 6 to 546 ± 35 ms; P <0.05) and elicited EADs and TdP that were suppressed by Ran (10 μM; n = 7 of 7 hearts). Simulations (Shannon but not Mahajan model) closely reproduced experimental data except for EAD suppression by Ran. Ran reduced open probability (P(o)) of RyR2 (half maximal inhibitory concentration = 10 ± 3 μM; n = 7) in bilayers and shifted half maximal effective concentration for Ca(2+)-dependent ryanodine binding from 0.42 ± 0.02 to 0.64 ± 0.02 μM with 30 μM Ran. CONCLUSIONS Ran reduces P(o) of RyR2, desensitizes Ca(2+)-dependent RyR2 activation, and inhibits Ca(i) oscillations, which represents a novel mechanism for its suppression of EADs and TdP.

Sulfhydryl oxidation and Ca2+ release from sarcoplasmic reticulum

SummaryOur interest in the role of sulfhydryl groups (SH) in regulating or altering transport across biological membranes has focused on the significance of a critical SH group associated with the Ca2+-release protein from skeletal muscle sarcoplasmic reticulum (SR). We have shown that binding of heavy metals to this group or oxidation of this sulfhydryl to a disulfide induces rapid Ca2+ release from SR vesicles [1, 2] and induces contraction in skinned muscle fibers [3]. Several models are described in which oxidation and reduction might control the state of the Ca2+-release channel from SR.

Rose Bengal activates the Ca2+ release channel from skeletal muscle sarcoplasmic reticulum

The photooxidizing xanthene dye rose bengal (10 nM to 1 microM) stimulates rapid Ca2+ release from skeletal muscle sarcoplasmic reticulum vesicles. Following fusion of sarcoplasmic reticulum (SR) vesicles to an artificial bilayer, reconstituted Ca2+ channel activity is stimulated by nanomolar concentrations of rose bengal in the presence of a broad-spectrum light source. Rose bengal does not appear to affect K+ channels present in the SR. Following reconstitution of the sulfhydryl-activated 106-kDa Ca2+ channel protein into a bilayer, rose bengal activates the isolated protein in a light-dependent manner. Ryanodine at a concentration of 10 nM is shown to lock the 106-kDa channel protein in a subconductance state which can be reversed by subsequent addition of 500 nM rose bengal. This apparent displacement of bound ryanodine by nanomolar concentrations of rose bengal is also directly observed upon measurement of [3H]ryanodine binding to JSR vesicles. These observations indicate that photooxidation of rose bengal causes a stimulation of the Ca2+ release protein from skeletal muscle sarcoplasmic reticulum by interacting with the ryanodine binding site. Furthermore, similar effects of rose bengal on isolated SR vesicles, on single channel measurements following fusion of SR vesicles, and following incorporation of the isolated 106-kDa protein strongly implicates the 106-kDa sulfhydryl-activated Ca2+ channel protein in the Ca2+ release process.

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

SummaryThe 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 μm). 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 μm), A 23187 (100 μg 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.