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首页> 外文期刊>Cellular Physiology and Biochemistry >Role for SUR2A in Coupling Cardiac KATP Channels to Caveolin-3
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Role for SUR2A in Coupling Cardiac KATP Channels to Caveolin-3

机译：SUR2A在将心脏KATP通道偶联到Caveolin-3中的作用

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ATP-sensitive Ksup+/sup channels in the heart are localized in the caveolae. However, little is known about the molecular mechanism underlying the caveolar targeting of those ion channels. The present study was designed to explore the molecular compositions involved in the interaction between cardiac KsubATP/sub channels and caveolin-3. The HA-tagged wild-type (Kir6.2/SUR2A) or mutant KsubATP/sub channel (Kir6.2C4A or Kir6.2Δ36) subunits were transiently transfected into COS-7 cells with or without caveolin-3. Both Kir6.2C4A and Kir6.2Δ36 are able to form tetrameric Kir6.2 channels on the cell membrane without SUR subunit. We demonstrated that caveolin-3 co-immunopreciptiated Kir6.2 in COS-7 cells transfected with Kir6.2/SUR2A/caveolin-3, whereas Kir6.2 was not detected in the caveolin-3 immunoprecipitates in cells transfected with either caveolin-3 or Kir6.2/SUR2A alone. In cells transfected with Kir6.2C4A/caveolin-3, Kir6.2C4A was detected with anti-HA antibody but at a significantly lower level in the caveolin-3 immunoprecipitates when compared with Kir6.2 detected in cells transfected with the Kir6.2/SUR2A/caveolin-3. Kir6.2C4A was not co-immunoprecipitated with caveolin-3 in cells transfected with caveolin-3 or Kir6.2C4A alone. The application of caveolin-3 scaffolding domain peptide, corresponding to amino acid residues 55-74 of caveolin-3, largely blocked the co-immunoprecipitation of caveolin-3 and Kir6.2/SUR2A octameric channels but did not prevent the co-immunoprecipitation of caveolin-3 and the Kir6.2 tetrameric channels. Disrupting caveolae with methyl-β-cyclodextrin significantly attenuated association of tetrameric Kir6.2 channels with caveolin-3. Immunofluorescence microscopy revealed that a higher percentage of cells showed significant colocalization of caveolin-3 with Kir6.2 than colocalization of caveolin-3 with Kir6.2C4A or Kir6.2Δ36. We further confirmed that in adult rat cardiac myocytes the association of endogenous octameric KsubATP/sub channels with caveolin-3 was largely prevented by caveolin-3 scaffolding domain peptide but not control peptide. We concluded that SUR2A is important for coupling cardiac KsubATP/sub channels to caveolin-3, possibly through the caveolin-3 scaffolding domain.
机译：心脏中对ATP敏感的K + 通道位于小窝中。然而，关于这些离子通道的腔穴靶向的分子机制知之甚少。本研究旨在探讨参与心脏K ATP 通道与小窝蛋白3相互作用的分子组成。将带有HA标记的野生型（Kir6.2 / SUR2A）或突变的K ATP 通道（Kir6.2C4A或Kir6.2Δ36）亚基瞬时转染到带有或不带有Caveolin-3的COS-7细胞中。 Kir6.2C4A和Kir6.2Δ36都能够在没有SUR亚基的细胞膜上形成四聚体Kir6.2通道。我们证明了在用Kir6.2 / SUR2A / caveolin-3转染的COS-7细胞中，caveolin-3共免疫沉淀了Kir6.2，而在用Caveolin-3转染的细胞中，在Caveolin-3免疫沉淀中未检测到Kir6.2。或单独使用Kir6.2 / SUR2A。在用Kir6.2C4A / caveolin-3转染的细胞中，用抗HA抗体检测到Kir6.2C4A，但与用Kir6.2 / 4转染的细胞中检测到的Kir6.2相比，在Caveolin-3免疫沉淀物中的检测到的水平显着降低。 SUR2A / caveolin-3。在单独用caveolin-3或Kir6.2C4A转染的细胞中，Kir6.2C4A未与caveolin-3共免疫沉淀。对应于caveolin-3氨基酸残基55-74的caveolin-3支架结构域肽的应用在很大程度上阻断了caveolin-3和Kir6.2 / SUR2A八聚体通道的免疫共沉淀，但并未阻止cavolin-3和Kir6.2 / SUR2A八聚体通道的免疫共沉淀。小窝蛋白3和Kir6.2四聚体通道。用甲基-β-环糊精破坏小窝可显着减弱四聚体Kir6.2通道与小窝3的缔合。免疫荧光显微镜检查显示，与cavolin-3与Kir6.2C4A或Kir6.2Δ36的共定位相比，较高百分比的细胞显示caveolin-3与Kir6.2的显着共定位。我们进一步证实，在成年大鼠心肌细胞中，caveolin-3支架结构域肽而非对照肽在很大程度上阻止了内源性八聚体K ATP 通道与Caveolin-3的结合。我们得出的结论是，SUR2A对于将心脏K ATP 通道（可能通过caveolin-3支架结构域）偶联至caveolin-3非常重要。

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