Reactive disulfides trigger Ca2+ release from sarcoplasmic reticulum via an oxidation reaction

N F Zaidi,C F Lagenaur,J J Abramson,I Pessah,G Salama

doi:10.1016/s0021-9258(20)88246-5

N F Zaidi, C F Lagenaur + Show 3 more

Open Access

https://doi.org/10.1016/s0021-9258(20)88246-5

Copy DOI

Abstract

Reactive disulfide compounds (RDSs) with a pyridyl ring adjacent to the S-S bond such as 2,2'-dithiodipyridine (2,2'-DTDP), 4,4'-dithiodipyridine, and N-succinimidyl 3(2-pyridyldithio)propionate (SPDP) trigger Ca2+ release from sarcoplasmic reticulum (SR) vesicles. They are known to specifically oxidize free SH sites via a thiol-disulfide exchange reaction with the stoichiometric production of thiopyridone. Thus, the formation of a mixed S-S bond between an accessible SH site on an SR protein and a RDS causes large increases in SR Ca2+ permeability. Reducing agents, glutathione (GSH) or dithiothreitol reverse the effect of RDSs and permit rapid re-uptake of Ca2+ by the Ca2+, Mg2+-ATPase. The RDSs, 2,2'-DTDP, 4,4'-dithiodipyridine and SPDP displaced [3H]ryanodine binding to the Ca2+-receptor complex at IC50 values of 7.5 +/- 0.2, 1.5 +/- 0.1, and 15.4 +/- 0.1 microM, respectively. RDSs did not alter the rapid initial phase of Ca2+ uptake by the pump, stimulated ATPase activity, and induced release from passively loaded vesicles with nonactivated pumps; thus they act at a Ca2+ release channel and not at the Ca2+, Mg2+-ATPase. Efflux rates increased in 0.25-1.0 mM [Mg2+]free then decreased in 2-5 mM [Mg2+]free. Adenine nucleotides inhibited the oxidation of SHs on SR protein by RDSs and thus reduced Ca2+ efflux rates. However, once RDSs oxidized these SH sites and opened the Ca2+ release pathway, subsequent additions of nucleotides stimulated Ca2+ efflux. In skinned fibers, 2,2'-dithiodipyridine elicited rapid twitches which were blocked by ruthenium red. These results indicate that RDSs trigger Ca2+ release from SR by oxidizing a critical SH group, and thus provide a method to covalently label the protein(s) involved in causing these changes in Ca2+ permeability.

Highlights

Reactive disulfidecompounds (RDSs) with a pyridyl The propagation of an action potential along transversering adjacent to theS-S bond such as 2,2’-dithiodipyr- tubular membranes in skeletal muscle triggers the release of idine (2,2’-DTDP), 4,4’-dithiodipyridine, and N-suc- Ca2+from the sarcoplasmicreticulum (SR)’ networkand cinimidyl 3(2-pyridyldithio)propionate (SPDP)trig- thereby initiates a contraction [1,2,3]
2-5 mM [Mg2+]freeA. denine nucleotides inhibited the permeable pore with a large unitary conductance which was oxidation of SHs on SR protein by RDSs and blocked by micromolar ruthenium red and millimolar M e reduced Ca2+efflux rates
These results indictahteat RDSs trigger Ca2+ release from SRby oxidizing a critical SH group,and provide a method tocovalentlylabel theprothe 400-450 kDa receptor is the “physiological” SR release channel since similar properties areobserved for Ca2+ release from skinned fibers [6, 20], heavy SR vesicles [21, 22], or “native” SR Ca2+channels incorporatedby fusing SR vesicles with planarbilayers [23,24,25]

Summary

RESULTS

Reactive Disulfides Induce SR Ca2+Release-RDS reagents are known to be absolutely specific to free SH groups and to readily oxidize S H groups at low substrate concentrations and high reaction rates [35]. The most likely thiol-disulreleased 50%of total releasable Ca2+.Disulfideswith a pyridyl fide exchange reactions between a free SH on an SR protein ring immediately adjacent to the S-S bond

Iloor A

DISCUSSION

Loading phase

Furtherexperimentsare needed to elucidate the possible role