Hart J D, Dulhunty A F
Muscle Research Group, John Curtin School of Medical Research, Australian National University, PO Box 334, Canberra, ACT, 2601, Australia.
J Membr Biol. 2000 Feb 1;173(3):227-36. doi: 10.1007/s002320001022.
We show that rabbit skeletal RyR channels in lipid bilayers can be activated or inhibited by NO, in a manner that depends on donor concentration, membrane potential and the presence of channel agonists. 10 microm S-nitroso-N-acetyl-penicillamine (SNAP) increased RyR activity at -40 mV within 15 sec of addition to the cis chamber, with a 2-fold increase in frequency of channel opening (F(o)). 10 microm SNAP did not alter activity at +40 mV and did not further activate RyRs previously activated by 2 mm cis ATP at +40 or -40 mV. In contrast to the increase in F(o) with 10 microm SNAP, 1 mm SNAP caused a 2-fold reduction in F(o) but a 1.5-fold increase in mean open time (T(o)) at -40 mV in the absence of ATP. 1 mm SNAP or 0.5 mm sodium nitroprusside (SNP) induced approximately 3-fold reductions in F(o) and T(o) at +40 or -40 mV when channels were activated by 2 mm cis ATP or in channels activated by 6.5 microm peptide A at -40 mV (peptide A corresponds to part of the II-III loop of the skeletal dihydropyridine receptor). Both SNAP-induced activation and SNAP/SNP-induced inhibition were reversed by 2 mm dithiothreitol. The results suggest that S-Nitrosylation or oxidation of at least three classes of protein thiols by NO each produced characteristic changes in RyR activity. We propose that, in vivo, initial release of NO activates RyRs, but stronger release increases [NO] and inhibits RyR activity and contraction.
我们发现,脂质双分子层中的兔骨骼肌兰尼碱受体(RyR)通道可被一氧化氮(NO)激活或抑制,其方式取决于供体浓度、膜电位以及通道激动剂的存在。向顺式腔室中添加10微摩尔的S-亚硝基-N-乙酰青霉胺(SNAP)后15秒内,可使-40 mV时的RyR活性增加,通道开放频率(F(o))增加2倍。10微摩尔的SNAP在+40 mV时不改变活性,并且在+40或-40 mV时不会进一步激活先前由2毫摩尔顺式ATP激活的RyR。与10微摩尔SNAP使F(o)增加相反,在无ATP的情况下,1毫摩尔SNAP在-40 mV时使F(o)降低2倍,但平均开放时间(T(o))增加1.5倍。当通道由2毫摩尔顺式ATP激活或在-40 mV时由6.5微摩尔肽A激活(肽A对应于骨骼肌二氢吡啶受体II-III环的一部分)时,1毫摩尔SNAP或0.5毫摩尔硝普钠(SNP)在+40或-40 mV时可使F(o)和T(o)降低约3倍。SNAP诱导的激活以及SNAP/SNP诱导的抑制均可被2毫摩尔二硫苏糖醇逆转。结果表明,NO对至少三类蛋白质硫醇的S-亚硝基化或氧化各自产生了RyR活性的特征性变化。我们提出,在体内,NO的初始释放激活RyR,但更强的释放会增加[NO]并抑制RyR活性和收缩。