Suko J, Hellmann G
Institute of Pharmacology, University of Vienna, Waehringerstrasse 13a, A-1090 Vienna, Austria.
Biochim Biophys Acta. 1998 Sep 16;1404(3):435-50. doi: 10.1016/s0167-4889(98)00075-5.
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 microM) increased the mean open probability (Po) of the calcium channel at subactivating (20 nM), maximally activating (20-100 microM 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 microM 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 microM 4-CMPS/p-CMB (Hill coefficient 1.7-2.0) at 4 microM Ca2+ and reduced at high concentrations (50-200 microM). The increase in [3H]ryanodine binding by 10 microM 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 microM 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.
通过使用纯化的钙释放通道进行单通道记录、放射性配体与肌浆网囊泡（高速离心肌浆网，HSR）结合以及HSR的钙释放，测定了两种有机汞化合物，4-（氯汞基）苯磺酸（4-CMPS）和对氯汞苯甲酸（p-CMB）对兔骨骼肌钙释放通道（雷诺丁受体）的作用。p-CMB或4-CMPS（20 - 100微摩尔）在亚激活（20纳摩尔）、最大激活（20 - 100微摩尔）和抑制（1 - 4毫摩尔）钙浓度下增加了钙通道的平均开放概率（Po），而对单位电导无影响。这种激活作用部分可被2毫摩尔二硫苏糖醇（DTT）逆转。两种化合物仅从胞质侧影响通道，而不从反侧影响。100微摩尔4-CMPS使Po短暂增加，随后在1分钟内进入低活性状态。在抑制性钙浓度下，Po增加到最大激活钙浓度或更低抑制性钙浓度时观察到的值。p-CMB/4-CMPS修饰的通道对雷诺丁敏感，并被钌红阻断。在4微摩尔钙浓度下，3 - 15微摩尔4-CMPS/p-CMB使[3H]雷诺丁结合增加至四倍（希尔系数1.7 - 2.0），而在高浓度（50 - 200微摩尔）下降低。10微摩尔4-CMPS引起的[3H]雷诺丁结合增加被2毫摩尔DTT完全抑制。4-CMPS显著增加了对特异性[3H]雷诺丁结合的高亲和力钙激活位点的亲和力，并降低了低亲和力钙抑制位点的亲和力。4-CMPS增加了雷诺丁受体对高亲和力雷诺丁结合的亲和力，而受体密度无变化。4-CMPS在亚激活钙浓度（20纳摩尔）下诱导被动钙负载的HSR囊泡快速、浓度依赖性、双相性钙释放，这一过程部分被4毫摩尔DTT抑制，被20微摩尔钌红完全阻断。有人提出，4-CMPS诱导的参与钙释放通道门控的必需巯基的调节导致了钙释放通道激活的高亲和力和抑制的低亲和力钙结合位点的表观钙亲和力的调节。
