Now in Center for Neuroscience & Regeneration/Rehabilitation Research, Department of Neurology, Yale University School of Medicine & Veterans Affairs Connecticut Healthcare System, USA.
Department of Biology, Georgia State University, Atlanta, GA 30302-4010, USA.
Acta Pharmacol Sin. 2014 Jan;35(1):58-64. doi: 10.1038/aps.2013.122. Epub 2013 Oct 14.
Highly reactive carbonyl methylglyoxal (MGO) is one of the metabolites excessively produced in diabetes. We have showed that prolonged exposure of vascular smooth muscle cells to MGO leads to instability of the mRNA encoding ATP-sensitive potassium (KATP) channel. In the present study we investigated the effects of MGO on the activity of KATP channels.
Kir6.1/ SUR2B, Kir6.2/SUR2B or Kir6.2Δ36 (a truncated Kir6.2 isoform) alone was expressed in HEK293 cells. Whole-cell currents were recorded in the cells with an Axopatch 200B amplifier. Macroscopic currents and single-channel currents were recorded in giant inside-out patches and normal inside-out patches, respectively. Data were analyzed using Clampfit 9 software.
The basal activity of Kir6.1/SUR2B channels was low. The specific KATP channel opener pinacidil (10 μmol/L) could fully activate Kir6.1/SUR2B channels, which was inhibited by the specific KATP channel blocker glibenclamide (10 μmol/L). MGO (0.1-10 mmol/L) dose-dependently activated Kir6.1/SUR2B channels with an EC50 of 1.7 mmol/L. The activation of Kir6.1/SUR2B channels by MGO was reversible upon washout, and could be inhibited completely by glibenclamide. Kir6.2Δ36 channels expressed in HEK293 cells could open automatically, and the channel activity was enhanced in the presence of MGO (3 mmol/L). Single channel recordings showed that MGO (3 mmol/L) markedly increased the open probability of Kir6.1/SUR2B channels, leaving the channel conductance unaltered.
Acute application of MGO activates KATP channels through direct, non-covalent and reversible interactions with the Kir6 subunits.
高度反应性羰基甲基乙二醛（MGO）是糖尿病中过度产生的代谢物之一。我们已经表明，血管平滑肌细胞长时间暴露于 MGO 会导致编码三磷酸腺苷敏感钾（KATP）通道的 mRNA 不稳定。在本研究中，我们研究了 MGO 对 KATP 通道活性的影响。
在 HEK293 细胞中单独表达 Kir6.1/SUR2B、Kir6.2/SUR2B 或 Kir6.2Δ36（一种截断的 Kir6.2 同工型）。使用 Axopatch 200B 放大器在细胞中记录全细胞电流。在巨内面向外斑和正常内面向外斑中分别记录宏观电流和单通道电流。使用 Clampfit 9 软件分析数据。
Kir6.1/SUR2B 通道的基础活性较低。特异性 KATP 通道 opener 吡那地尔（10 μmol/L）可完全激活 Kir6.1/SUR2B 通道，该通道被特异性 KATP 通道阻滞剂格列本脲（10 μmol/L）抑制。MGO（0.1-10 mmol/L）剂量依赖性地激活 Kir6.1/SUR2B 通道，EC50 为 1.7 mmol/L。MGO 引起的 Kir6.1/SUR2B 通道激活可在洗脱后逆转，并可被格列本脲完全抑制。在 HEK293 细胞中表达的 Kir6.2Δ36 通道可自动开放，并且在存在 MGO（3 mmol/L）时通道活性增强。单通道记录显示，MGO（3 mmol/L）显着增加 Kir6.1/SUR2B 通道的开放概率，而不改变通道电导。
急性应用 MGO 通过与 Kir6 亚基的直接、非共价和可逆相互作用激活 KATP 通道。
