Hughes B A, Takahira M
Departments of Ophthalmology and Physiology, University of Michigan, Ann Arbor, Michigan 48105, USA.
Am J Physiol. 1998 Nov;275(5):C1372-83. doi: 10.1152/ajpcell.1998.275.5.C1372.
Inwardly rectifying K+ current (IKir) in freshly isolated bovine retinal pigment epithelial (RPE) cells was studied in the whole cell recording configuration of the patch-clamp technique. When cells were dialyzed with pipette solution containing no ATP, IKir ran down completely in <10 min [half time (t1/2) = 1.9 min]. In contrast, dialysis with 2 mM ATP sustained IKir for 10 min or more. Rundown was also prevented with 4 mM GTP or ADP. When 0.5 mM ATP was used, IKir ran down by approximately 71%. Mg2+ was a critical cofactor because rundown occurred when the pipette solution contained 4 mM ATP but no Mg2+ (t1/2 = 1.8 min). IKir also ran down when the pipette solution contained 4 mM Mg2+ + 4 mM 5'-adenylylimidodiphosphate (t1/2 = 2.7 min) or 4 mM adenosine 5'-O-(3-thiotriphosphate) (t1/2 = 1.9 min), nonhydrolyzable and poorly hydrolyzable ATP analogs, respectively. We conclude that the sustained activity of IKir in bovine RPE requires intracellular MgATP and that the underlying mechanism may involve ATP hydrolysis.
采用膜片钳技术的全细胞记录模式，对新鲜分离的牛视网膜色素上皮（RPE）细胞中的内向整流钾电流（IKir）进行了研究。当用不含ATP的移液管溶液透析细胞时，IKir在不到10分钟内完全衰减[半衰期（t1/2）=1.9分钟]。相比之下，用2 mM ATP透析可使IKir维持10分钟或更长时间。4 mM GTP或ADP也可防止电流衰减。当使用0.5 mM ATP时，IKir衰减约71%。Mg2+是关键的辅助因子，因为当移液管溶液含有4 mM ATP但不含Mg2+时会发生电流衰减（t1/2 = 1.8分钟）。当移液管溶液含有4 mM Mg2+ + 4 mM 5'-腺苷酰亚胺二磷酸（t1/2 = 2.7分钟）或4 mM腺苷5'-O-（3-硫代三磷酸）（t1/2 = 1.9分钟）时，IKir也会衰减，这两种分别是不可水解和难水解的ATP类似物。我们得出结论，牛RPE细胞中IKir的持续活性需要细胞内MgATP，其潜在机制可能涉及ATP水解。
