The interaction of nucleotides with the tolbutamide block of cloned ATP-sensitive K+ channel currents expressed in Xenopus oocytes: a reinterpretation
The interaction of nucleotides with the tolbutamide block of cloned ATP-sensitive K+ channel currents expressed in Xenopus oocytes: a reinterpretation
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DOI：
10.1111/j.1469-7793.1997.00035.x
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发表时间：
1997-10-01
影响因子：
5.5
通讯作者：
Ashcroft, FM
中科院分区：
文献类型：
--
作者：
Gribble, FM;Tucker, SJ;Ashcroft, FM
1.利用在非洲爪哇卵母细胞中异源表达的野生型和突变型K-ATP通道，我们研究了核苷酸调节甲苯丁胺阻断β细胞ATP敏感性钾通道(K-ATP通道)的机制。该通道由磺酰基脲受体(SUR1)和造孔剂(Kir6.2)亚基组成。在无核苷酸条件下，甲苯丁胺阻断野生型K-ATP电流的量效关系表现为高亲和力(K-I=2.0µM)和低亲和力(K-I=1.8mAT)。Kir6.2 Delta C36(Kir6.2的截断形式，独立于SUR1表达)的甲苯丁胺阻滞剂的剂量-反应关系最适合于单一的低亲和力位点(K-I=1.7 mm)。这表明高亲和力位点位于SUR1上，而低亲和力位点位于Kir6.2.4上。ADP(100 MU M)对野生型K-ATP电流具有双重作用：该核苷酸在有镁离子存在时增强电流，在无镁离子存在时抑制电流。在有镁离子存在的情况下，100 mU M ADP可阻断Kir6.2 Delta C36电流。对于野生型K-ATP电流，在100 mU M镁ADP存在时，0.5 nM甲苯磺丁胺的阻断作用(84+/-2%)大于不存在时(59+/-4%)。当SUR1突变为取消K-ATP电流(K719A或K1384M)的镁ADP激活时，有无镁ADP.6对甲苯磺丁胺的抑制程度没有差别。对于野生型和K719A-K1384M电流，甲苯丁胺与高亲和力或低亲和力位点相互作用的K-I均不受100亩M-MgADP的影响。镁离子通道阻断剂(100 MU M)可增强野生型K-ATP电流，但对K719A-K1384M电流无明显影响。无论是在高亲和力部位还是低亲和力部位，它都不影响甲苯丁胺阻断的K-I。我们的结果表明，甲苯丁酰胺与SUR1上的高亲和力位点的相互作用取消了镁ADP的刺激作用。这揭示了ADP的抑制作用，并导致通道抑制明显增加。在生理条件下，取消镁ADP的激活可能是甲苯丁胺抑制K-ATP通道的主要机制。
1. We have examined the mechanism by which nucleotides modulate the tolbutamide block of the beta-cell ATP-sensitive K+ channel (K-ATP channel), using wild-type and mutant K-ATP channels heterologously expressed in Xenopus oocytes. This channel is composed of sulphonylurea receptor (SUR1) and pore-forming (Kir6.2) subunits.2. The dose-response relation for tolbutamide block of wild-type K-ATP currents in the absence of nucleotide showed both a high-affinity (K-i = 2.0 mu M) and a low-affinity (K-i = 1.8 mat) site.3. The dose-response relation for tolbutamide block of Kir6.2 Delta C36 (a truncated form of Kir6.2 which is expressed independently of SUR1) was best fitted with a single, low-affinity site (K-i = 1.7 mM). This indicates that the high-affinity site resides on SUR1, whereas the low-affinity site is located on Kir6.2.4. ADP (100 mu M) had a dual effect on wild-type K-ATP currents: the nucleotide enhanced the current in the presence of Mg2+, but was inhibitory in the absence of Mg2+. Kir6.2 Delta C36 currents were blocked by 100 mu M ADP in the presence of Mg2+.5. For wild-type K-ATP currents, the blocking effect of 0.5 nM tolbutamide appeared greater in the presence of 100 mu M MgADP (84 +/- 2%) than in its absence (59 +/- 4%). When SUR1 was mutated to abolish MgADP activation of K-ATP currents (K719A or K1384M), there was no difference in the extent of tolbutamide inhibition in the presence or absence of MgADP.6. The K-i for tolbutamide interaction with either the high- or low-affinity site was unaffected by 100 mu M MgADP, for both wild-type and K719A-K1384M currents.7. MgGDP (100 mu M) enhanced wild-type K-ATP currents and was without effect on K719A-K1384M currents. It did not affect the K-i for tolbutamide block at either the high-or low-affinity site.8. Our results indicate that interaction of tolbutamide with the high-affinity site (on SUR1) abolishes the stimulatory action of MgADP. This unmasks the inhibitory effect of ADP and leads to an apparent increase in channel inhibition. Under physiological conditions, abolition of MgADP activation is likely to constitute the principal mechanism by which tolbutamide inhibits the K-ATP channel.