Modification of the gating of the cardiac sarcoplasmic reticulum Ca(2+)-release channel by H2O2 and dithiothreitol

Abstract

The effect of hydrogen peroxide (H2O2) on the sheep cardiac sarcoplasmic reticulum (SR) Ca(2+)-release channel has been investigated under voltage-clamp conditions after incorporation of native membrane vesicles into planar phospholipid bilayers. In the presence of micromolar activating calcium concentrations on the cytosolic side of the membrane, H2O2 (3-5 mM) increased open probability of the channels. H2O2 did not affect the conductance of the channel or the response to activating compounds, such as ATP and caffeine. H2O2 did not alter the inhibitory response to magnesium or the modification of channels by ryanodine. At subactivating calcium concentrations (approximately 45 pM) on the cytosolic side of the membrane, 5 mM H2O2 was still able to open the channel. Analysis of single-channel open and closed lifetimes suggested that H2O2 had a direct effect on the gating mechanism of the channel. Open probability of the SR Ca(2+)-release channel is reduced by millimolar concentrations of dithiothreitol, a sulfhydryl-protecting compound, in a concentration-dependent manner. In conclusion, it is probable that H2O2 activates the SR Ca(2+)-release channel via an oxidation of cysteine thiol groups in the channel protein.

Ryanodine Receptors: Structure, Expression, Molecular Details, and Function in Calcium Release

“…RyR is an established redox-sensitive channel and alterations in its redox state can result in either activation (Stoyanovsky et al 1997;Eager and Dulhunty 1998) or inactivation (Boraso and Williams 1994;Marengo et al 1998). Other key components of Ca 2þ regulation and E-C coupling, e.g., SERCA and Ca v 's, are also redox modulated.…”

The effect of oxygen free radicals on calcium current and dihydropyridine binding sites in guinea‐pig ventricular myocytes

“…We and other groups have shown in guinea-pig and rat isolated myocytes that these changes are the consequence of intracellular calcium loading (Beresewicz & Horackova, 1991;Cerbai et al, 1991;Jabr & Cole, 1993;Mugelli et al, 1995), possibly involving abnormalities in sarcoplasmic reticulum Ca2+ release (Shattock et al, 1990;Boraso & Williams, 1994). In fact, prolongation of action potential duration and development of early-(EAD) and delayed afterdepolarizations (DAD) are prevented by intracellular EGTA (Cerbai et al, 1991;Jabr & Cole, 1993;Mugelli et al, 1995) and/or by pretreatment of the cells with ryanodine (Beresewics & Horachova, 1991).…”

Multiple Classes of Sulfhydryls Modulate the Skeletal Muscle Ca2+ Release Channel

“…The channel opens in response to a signal from the transverse tubules, which is triggered by membrane depolarization (2), and the resulting flux of Ca 2ϩ from the sarcoplasmic reticulum initiates the sequence of events that leads to muscle contraction. Several laboratories have clearly demonstrated that the activity of the Ca 2ϩ release channel is modulated by oxidation-reduction reactions (3)(4)(5)(6)(7)(8)(9)(10)(11). Oxidizing agents that stimulate Ca 2ϩ release from the sarcoplasmic reticulum include H 2 O 2 (3), 2,2Ј-dithiodipyridine, 4,4Ј-dithiodipyridine (4), Cu 2ϩ phthalocyanine dyes (5), anthraquinone doxorubicin (6,7), and thimerosal (8).…”

“…These studies led Abramson and Salama (10) to propose a model for redox modulation of the channel that involves three different sulfhydryl groups that exist in close proximity and that can form mixed disulfides to open or close the channel. The evidence that the channel can be altered by oxidation is conclusive (3)(4)(5)(6)(7)(8)(9)(10)(11); the question is whether this oxidation plays a physiological role in skeletal muscle. It is not yet known if disulfide interchange or oxidation-reduction of sulfhydryls on the Ca 2ϩ release channel contribute to normal excitation-contraction coupling, but an increasing body of evidence suggests that such a mechanism could be an important modulatory element.…”