The present study demonstrates the following characteristic suramin actions on the purified skeletal muscle calcium release channel in single-channel current recordings and [(3)H]ryanodine binding to HSR: 1) Suramin (0.3-0.9 mM) induced a concentration-dependent increase in the open probability (P(o) congruent with 0.9) at 20 to 100 microM Ca(2+) and an almost fully open channel at 1 mM Ca(2+) (P(o) = 0.95) with a marked shift to longer open states (tau(o)3/tau(o)4). Suramin increased the apparent calcium affinity to the activating high-affinity calcium binding sites and reduced the apparent magnesium affinity to the inhibitory low affinity Ca(2+)/Mg(2+) binding sites. 2) Channel activation by suramin and sulfhydryl oxidation was additive. 3) Suramin (0.9 mM) reversed the Ca-calmodulin-induced channel inhibition at 0.1 or 1 to 5 microM Ca-calmodulin. 4) The open probability of the suramin activated channel was almost completely inhibited by 10 mM Mg(2+) or Ca(2+) on short suramin exposure. Prolonged suramin exposure (30-60 min) resulted in a time-dependent, slow increase in P(o), with long open states of low frequency in the presence of 10 to 20 mM Mg(2+) or Ca(2+). 5) Magnesium induced inhibition of P(o) (IC(50) = 0.38 mM) and equilibrium [(3)H]ryanodine binding (IC(50) = 0.30 mM) agreed well in control channels, but were dissociated in the presence of 0.9 to 1.0 mM suramin (IC(50) = 0.82 mM versus 83 mM). [(3)H]ryanodine binding seemed to monitor predominantly the long-term alteration in channel function. 6) The multiple effects of suramin on channel function suggest an allosteric mechanism and no direct effects on binding of endogenous ligands involved in channel gating.

The modulation of the calmodulin-induced inhibition of the calcium release channel (ryanodine receptor) by two sulfhydryl oxidizing compounds, 4-(chloromercuri)phenyl–sulfonic acid (4-CMPS) and 4,4′-dithiodipyridine (4,4′-DTDP) was determined by single channel current recordings with the purified and reconstituted calcium release channel from rabbit skeletal muscle sarcoplasmic reticulum (HSR) and [3H]ryanodine binding to HSR vesicles. 0.1 μm CaM reduced the open probability (P o ) of the calcium release channel at maximally activating calcium concentrations (50–100 μm) from 0.502 ± 0.02 to 0.137 ± 0.022 (n= 28), with no effect on unitary conductance. 4-CMPS (10–40 μm) and 4,4′-DTDP (0.1–0.3 mm) induced a concentration dependent increase in P o (> 0.9) and caused the appearance of longer open states. CaM shifted the activation of the calcium release channel by 4-CMPS or 4,4′-DTDP to higher concentrations in single channel recordings and [3H]ryanodine binding. 40 μm 4-CMPS induced a near maximal (P o > 0.9) and 0.3 mm 4,4′-DTDP a submaximal (P o = 0.74) channel opening in the presence of CaM, which was reversed by the specific sulfhydryl reducing agent DTT. Neither 4-CMPS nor 4,4′-DTDP affected Ca-[125I]calmodulin binding to HSR. 1 mm MgCl2 reduced P o from 0.53 to 0.075 and 20–40 μm 4-CMPS induced a near maximal channel activation (P o > 0.9). These results demonstrate that the inhibitory effect of CaM or magnesium in a physiological concentration is diminished or abolished at high concentrations of 4-CMPS or 4,4′-DTDP through oxidation of activating sulfhydryls on cysteine residues of the calcium release channel.

The actions of the nitric oxide (NO) donors 1-hydroxy-2-oxo-3-(N-methyl-3-aminopropyl)-3 methyl-1-triazine (NOC-7), S-nitrosoacetylcysteine (CySNO) and S-nitrosoglutathione (GSNO) on the purified calcium release channel (ryanodine receptor) of rabbit skeletal muscle were determined by single channel current recordings. In addition, the activation of the NO donor modulated calcium release channel by the sulfhydryl oxidizing organic mercurial compound 4-(chloromercuri)phenylsulfonic acid (4-CMPS) was investigated. NOC-7 (0.1 and 0.3 mM) and CySNO (0.4 and 0.8 mM) increased the open probability (Po) of the calcium release channel at activating calcium concentrations (20–100 μM Ca2+) by 60–100%, with no effect on the current amplitude; this activation was abolished by the specific sulfhydryl reducing agent DTT. High concentrations of CySNO (1.6–2 mM) decreased Po. Activation by GSNO (1 mM) was observed in two thirds of the experiments, but 2 mM and 4 mM GSNO markedly reduced Po at activating Ca2+ (20–100 μM). In contrast to 4-CMPS, NOC-7 or GSNO had no effect at subactivating free Ca2+ (0.6 μM). 4-CMPS further increased the open probability of NOC-7- or CySNO-stimulated channels and reversed transiently the reduced open probability of CySNO or GSNO inhibited channels at activating free Ca2+. High concentrations of GSNO did not prevent channel activation of 4-CMPS at subactivating free Ca2+. The NOC-7-, CySNO- or GSNO-modified channels were completely blocked by ruthenium red. It is suggested that nitrosylation/oxidation of sulfhydryls by NO donors and oxidation of sulfhydryls by 4-CMPS affect different cysteine residues essential in the gating of the calcium release channel.

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 (Po) of the calcium channel at subactivating (20 nM), maximally activating (20–100 μM) and inhibitory (1–4 mM) Ca2+ 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 Po, followed by a low activity state within 1 min. At inhibitory Ca2+ concentrations Po was increased to values observed with maximally activating Ca2+ or lower, inhibitory Ca2+ 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 Ca2+ 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 Ca2+ 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.

1.1. The effect was studied of local anesthetics (tetracaine, dibucaine, procaine and xylocaine) on the forward and the backward reactions of the calcium pump of skeletal muscle sarcoplasmic reticulum.2.2. The inhibition of the rate of calcium uptake, the rate of calcium-dependent ATP splitting and the rate of calcium-dependent ATP-ADP phosphate exchange by sarcoplasmic reticulum in the presence of the above drugs is at least partially due to the inhibition of the phosphoprotein formation from ATP.3.3. The rate of the ADP-induced calcium release from sarcoplasmic reticulum and the rate of ATP synthesis driven by the calcium efflux are inhibited on account of a reduction of the phosphoprotein formation by orthophosphate.4.4. The phosphorylation of calcium transport ATPase by either ATP or orthophosphate is diminished by the local anesthetics owing to a reduction in the apparent calcium affinity of sarcoplasmic reticulum membranes on the outside and on the inside, respectively.5.5. The drug-induced calcium efflux from calcium-preloaded sarcoplasmic reticulum vesicles, a reaction not requiring ADP, is probably not mediated by calcium transport ATPase.