Mechanisms on the reperfusion arrythmias and the protection from reperfusion arrythmias : Redox reguration of the cardiac ion channels

为了阐明再灌注心律失常的机制，我们着重研究了氧化应激对心肌离子通道的作用。应用膜片钳技术研究了巯基氧化剂对培养的COS 7细胞心脏和骨骼肌Na通道α亚基（hH 1和hskm 1）表达的影响。1)巯基氧化剂对hskm1的影响：HgCl 2以剂量依赖的方式抑制hH 1，尽管硫柳汞或DTDP不能影响hskm1。DTT预处理或后处理均能保护hskm1免受HgCl_2的抑制。2）hH_1与hskm1对巯基氧化剂敏感性的比较。HgCl 2对hH1的抑制作用的Kd值显著低于对hskm1的抑制作用。根据Na通道a亚基的成孔区的氨基酸序列，结构域1的P环区的半胱氨酸存在于hill中，而不存在于hskm 1中。3）结构域1的P环区的半胱氨酸373的作用。为了阐明hill对HgCl_2敏感性增高的机理，我们在hill中引入了373位酪氨酸取代373位半胱氨酸的点突变，并研究了HgCl_2对突变hH_1的影响。该突变体获得了对HgCl 2的抗性。这些结果表明，氧化应激通过在半胱氨酸残基之间形成二硫键来阻断Na通道，半胱氨酸373在心脏对氧化应激的特异性敏感性中起重要作用。

To elucidate the mechanism on the reperfusion arrythmias, we focused on the role of oxidative stress on the cardiac ion channel. We studied the effects of sulfyhydryl oxidative agents on the expressed Na channel alpha subuit of heart (hH1) or skeletal muscle (hskm1) of the cultured COS7 cells using patch clamp techniques. 1) Effects of sulfhydyl oxidative agents on hskm1 : HgCI2 depressed hH1 in dose-depedent manner, although either thimerosal or DTDP could not affect hskm1. Either pretreatment or post treatment with DTT could protect hskm1 from inhibition by HgCl2.2) Comparison of the sensitivity of hH1 to sulfhydryl oxidative agents with that of hskm1. : The Kd of HgCl2 for inhibition of hH1 was significantly lower than that for inhibition of hskm1. According to the amino sequences of the pore forming region of the Na channel a subuit, the cysteine of the P loop region in the domain 1 was existing in hill not in hskm1.3) The role of the cysteine 373 in the p loop region of the domain 1. : To elucidate the mechansm on the higher sensitivity of hill to HgCl2, we introduced the point mutation substituting tyrosine 373 for cysteien 373 in hill and studied the effect of HgCl2 on mutant hHl. This mutant acquired the resistance against HgCl2. These results suggest that oxidative stress block Na channel via forming the disulfide bond among the cysteine residues and cysteine 373 played the povotal role for the heart specific sensitivity to oxidaitve stress.