Modification of sulfhydryls of the skeletal muscle calcium release channel by organic mercurial compounds alters Ca 2+ affinity of regulatory Ca 2+ sites in single channel recordings and [ 3H]ryanodine binding

Abstract

The actions of two organic mercurial compounds, 4-(chloromercuri)phenyl-sulfonic acid (4-CMPS) and p-chloromercuribenzoic acid ( p-CMB) on the calcium release channel (ryanodine receptor) from rabbit skeletal muscle were determined by single channel recordings with the purified calcium release channel, radioligand binding to sarcoplasmic reticulum vesicles (HSR) and calcium release from HSR. p-CMB or 4-CMPS (20–100 μM) increased the mean open probability ( P o) of the calcium channel at subactivating (20 nM), maximally activating (20–100 μM) and inhibitory (1–4 mM) Ca 2+ concentrations, with no effect on unitary conductance. This activation was partly reversed by 2 mM DTT. Both compounds affected the channels only from the cytosolic side, but not from the trans side. 100 μM 4-CMPS caused a transient increase in P o, followed by a low activity state within 1 min. At inhibitory Ca 2+ concentrations P o was increased to values observed with maximally activating Ca 2+ or lower, inhibitory Ca 2+ concentrations. The p-CMB/4-CMPS modified channels were ryanodine sensitive and blocked by ruthenium red. [ 3H]Ryanodine binding was increased up to four-fold with 3–15 μM 4-CMPS/ p-CMB (Hill coefficient 1.7–2.0) at 4 μM Ca 2+ and reduced at high concentrations (50–200 μM). The increase in [ 3H]ryanodine binding by 10 μM 4-CMPS was completely inhibited by 2 mM DTT. 4-CMPS significantly increased the affinity for the high affinity calcium activation sites and decreased the affinity of low affinity calcium inhibitory sites of specific [ 3H]ryanodine binding. 4-CMPS increased the affinity of the ryanodine receptor for high affinity ryanodine binding without a change in receptor density. 4-CMPS induced a rapid, concentration-dependent, biphasic calcium release from passively calcium-loaded HSR vesicles at subactivating Ca 2+ concentrations (20 nM), which was partly inhibited by 4 mM DTT and completely blocked by 20 μM ruthenium red. It is suggested that the 4-CMPS-induced modulation of essential sulfhydryls involved in the gating of the calcium release channel results in a modulation of the apparent calcium affinity of the activating high affinity and inhibitory low affinity calcium binding sites of the calcium release channel.