Molecular and functional analysis of ATP-sensitive K^+ channel on the nuclear envelope

本研究旨在检测心肌细胞核膜上是否存在ATP敏感性K^(K&lt；ATP&gt；)通道，以确定核膜上K&lt；ATP&gt；通道的功能分子及其生理作用。通过格列本脲-BODIPY-FL与磺脲受体的结合实验，我们检测了核膜上K&lt；ATP&gt；通道的存在。用共聚焦显微镜观察到格列本脲-BODIPY-FL与核标记物Hoechst的结合，提示心肌细胞核膜上存在磺脲受体。用膜片钳技术记录单个核膜的单通道活动。虽然从核膜上可以记录到几种单通道活动，但格列本脲、甲苯磺丁胺和吡那地尔不影响该通道的活动。用共聚焦显微镜和钙敏感的荧光染料Fluo-3/AM测量细胞核内的钙信号。格列本脲和甲苯丁脲对核膜钙离子浓度均无影响。因此，即使K_lt；ATP&gt；通道存在于心肌细胞的核膜上，该通道的密度或生理意义也可能不会那么大。

This study was undertaken to test whether ATP-sensitive K^+ (K_<ATP> ) channel exists on the nuclear envelope of cardiac cells and to determine the functional molecules and physiological roles of K_<ATP> channel on the nuclear envelope. Using binding assay of glibenclamide-BODIPY-FL to sulfonylurea receptor we tested the existence of K_<ATP> channel on the nuclear envelope. We could observe the binding of glibenclamide-BODIPY-FL colocalized with Hoechst, a nuclear marker, using confocal microscopy, suggesting the existence of sufonylurea receptor on the nuclear envelope of cardiomyocytes. The single channel activity was recorded from the nuclear envelope of isolated nuclei using patch clamp techniques. Although several kinds of single channel activity could be recorded from the nuclear envelope, the channel activity was not influenced by glibenclamide, tolbutamide, or pinacidil. Nuclear Ca^<2+> signal was measured using confocal microscopy and the Ca^<2+> sensitive fluorescence dye Fluo-3/AM. Neither glibenclamide nor tolbutamide affected Ca^<2+> concentration in the nuclear envelope. Thus, even if K_<ATP> channel exists on the nuclear envelope of cardiac cells, the density or physiological significance of the channel may not be so large.