Department of Pharmacology, University of Rochester Medical Center, 601 Elmwood Ave, Rochester, NY 14642, USA.
Circ Res. 2010 Apr 16;106(7):1190-6. doi: 10.1161/CIRCRESAHA.109.215400. Epub 2010 Feb 25.
The mitochondrial ATP sensitive potassium channel (mK(ATP)) is implicated in cardioprotection by ischemic preconditioning (IPC), but the molecular identity of the channel remains controversial. The validity of current methods to assay mK(ATP) activity is disputed.
We sought to develop novel methods to assay mK(ATP) activity and its regulation.
Using a thallium (Tl(+))-sensitive fluorophore, we developed a novel Tl(+) flux based assay for mK(ATP) activity, and used this assay probe several aspects of mK(ATP) function. The following key observations were made. (1) Time-dependent run down of mK(ATP) activity was reversed by phosphatidylinositol-4,5-bisphosphate (PIP(2)). (2) Dose responses of mK(ATP) to nucleotides revealed a UDP EC(50) of approximately 20 micromol/L and an ATP IC(50) of approximately 5 micromol/L. (3) The antidepressant fluoxetine (Prozac) inhibited mK(ATP) (IC(50)=2.4 micromol/L). Fluoxetine also blocked cardioprotection triggered by IPC, but did not block protection triggered by a mK(ATP)-independent stimulus. The related antidepressant zimelidine was without effect on either mK(ATP) or IPC.
The Tl(+) flux mK(ATP) assay was validated by correlation with a classical mK(ATP) channel osmotic swelling assay (R(2)=0.855). The pharmacological profile of mK(ATP) (response to ATP, UDP, PIP(2), and fluoxetine) is consistent with that of an inward rectifying K(+) channel (K(IR)) and is somewhat closer to that of the K(IR)6.2 than the K(IR)6.1 isoform. The effect of fluoxetine on mK(ATP)-dependent cardioprotection has implications for the growing use of antidepressants in patients who may benefit from preconditioning.
线粒体 ATP 敏感性钾通道（mK(ATP)）与缺血预处理（IPC）的心脏保护作用有关，但通道的分子身份仍存在争议。目前测定 mK(ATP) 活性的方法的有效性存在争议。
我们试图开发新的方法来测定 mK(ATP) 的活性及其调节。
使用铊（Tl(+)）敏感荧光探针，我们开发了一种新的基于 Tl(+) 通量的 mK(ATP) 活性测定法，并使用该测定探针研究了 mK(ATP) 功能的几个方面。得出以下关键观察结果。(1) mK(ATP) 活性的时间依赖性衰减可被磷脂酰肌醇-4,5-二磷酸（PIP(2)）逆转。(2) mK(ATP) 对核苷酸的剂量反应显示 UDP 的 EC(50)约为 20 微摩尔/L，而 ATP 的 IC(50)约为 5 微摩尔/L。(3) 抗抑郁药氟西汀（Prozac）抑制 mK(ATP)（IC(50)=2.4 微摩尔/L）。氟西汀还阻断了由 IPC 触发的心脏保护作用，但不阻断由 mK(ATP) 非依赖性刺激触发的保护作用。相关的抗抑郁药齐美定对 mK(ATP) 或 IPC 均无影响。
Tl(+) 通量 mK(ATP) 测定法通过与经典 mK(ATP) 通道渗透肿胀测定法的相关性得到验证（R(2)=0.855）。mK(ATP) 的药理学特征（对 ATP、UDP、PIP(2) 和氟西汀的反应）与内向整流钾通道（K(IR)）一致，与 K(IR)6.2 亚型更为接近，而与 K(IR)6.1 亚型有所不同。氟西汀对 mK(ATP) 依赖性心脏保护作用的影响对越来越多的在可能受益于预处理的患者中使用抗抑郁药具有重要意义。
