Modulation of recombinant human cardiac L-type Ca2+ channel α1C subunits by redox agents and hypoxia
Modulation of recombinant human cardiac L-type Ca2+ channel α1C subunits by redox agents and hypoxia
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DOI：
10.1111/j.1469-7793.1999.629ad.x
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发表时间：
1999-02-01
影响因子：
5.5
通讯作者：
Peers, C
中科院分区：
文献类型：
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作者：
Fearon, IM;Palmer, ACV;Peers, C
1.采用全细胞膜片钳技术研究还原剂和氧化剂对稳定表达于人胚肾（HEK 293）细胞的重组人心脏L型钙通道α（1C）亚基的调节作用.氧化剂硫柳汞（10 μ M）和p-氯汞苯磺酸（PCMBS; 2 μ M至2 mM）造成不可逆的抑制钙离子通道电流。还原剂1，4-二硫苏糖醇（DTT; 2 mM）对Ca 2+通道电流无影响，但可逆转硫柳汞和PCMBS的抑制作用. Ca 2+通道电流也被甲硫基磺酸盐化合物（2-氨基乙基）甲硫基磺酸盐（MTSEA，2.5 mM）预处理抑制，但不受（2-磺酸基乙基）甲硫基磺酸盐（MTSES，10 mM）相同的预处理。MTSEA的作用可被DTT（2 mM）完全逆转。由200 μ m PCMBS引起的电流抑制程度没有受到MTSEA预处理的显著影响，并且在PCMBS处理之后，MTSEA引起与先前未用PCMBS处理的细胞中观察到的抑制程度相似的抑制程度。这些结果表明，不同的巯基基团被这两种试剂调节。钙通道电流的抑制作用不受MTSEA预处理细胞的影响，但PCMBS治疗完全阻止。我们的结果表明，不同的半胱氨酸残基上的ct，亚基可以进行氧化还原调节，并在这样做改变通道功能。这些残基中的一些，但不是全部，似乎与缺氧抑制该通道的机制有关。
1. Whole-cell patch clamp recordings were used to investigate the modulation by reducing and oxidizing agents of recombinant human cardiac L-type Ca2+ channel alpha(1C) subunits stably expressed in human embryonic kidney (HEK 293) cells.2. The oxidizing agents thimerosal (10 mu M) and p-chloromercuribenzene sulphonic acid (PCMBS; 2 mu M to 2 mM) caused irreversible inhibition of Ca2+ channel currents. The reducing agent 1,4-dithiothreitol (DTT; 2 mM) was without effect on Ca2+ channel currents, but reversed the inhibitory actions of thimerosal and PCMBS.3. Ca2+ channel currents were also inhibited by pretreatment with the methanethiosulphonate compound (2-aminoethyl)methanethiosulphonate (MTSEA, 2.5 mM), but were unaffected by identical pretreatment with (2-sulphonatoethyl)methanethiosulphonate (MTSES, 10 mM). The effects of MTSEA could be fully reversed by DTT (2 mM). The degree of current inhibition caused by 200 mu m PCMBS was not significantly affected by pretreatment with MTSEA, and following PCMBS treatment, MTSEA caused a similar degree of inhibition to that observed in cells that were not previously treated with PCMBS. These findings suggested that distinct thiol groups were modulated by these two agents.4. Hypoxic inhibition of Ca2+ channel currents was unaffected by pretreatment of cells with MTSEA but was fully prevented by treatment with PCMBS. Our results indicate that distinct cysteine residues on the ct,, subunit can undergo redox modulation and in so doing alter channel function. Some, but not all, of these residues appear to be associated with the mechanism underlying inhibition of this channel by hypoxia.