Skatchkov Serguei N, Rojas Legier, Eaton Misty J, Orkand Richard K, Biedermann Bernd, Bringmann Andreas, Pannicke Thomas, Veh Rüdiger W, Reichenbach Andreas
CMBN, Department of Biochemistry, School of Medicine, Universidad Central del Caribe, Bayamón, Puerto Rico.
Glia. 2002 May;38(3):256-67. doi: 10.1002/glia.10073.
The retinae and brains of larval and adult amphibians survive long-lasting anoxia; this finding suggests the presence of functional K(ATP) channels. We have previously shown with immunocytochemistry studies that retinal glial (Müller) cells in adult frogs express the K(ATP) channel and receptor proteins, Kir6.1 and SUR1, while retinal neurons display Kir6.2 and SUR2A/B (Skatchkov et al., 2001a: NeuroReport 12:1437-1441; Eaton et al., in press: NeuroReport). Using both immunocytochemistry and electrophysiology, we demonstrate the expression of Kir6.1/SUR1 (K(ATP)) channels in adult frog and tadpole Müller cells. Using conditions favoring the activation of K(ATP) channels (i.e., ATP- and spermine-free cytoplasm-dialyzing solution containing gluconate) in Müller cells isolated from both adult frogs and tadpoles, we demonstrate the following. First, using the patch-clamp technique in whole-cell recordings, tolbutamide, a blocker of K(ATP) channels, blocks nearly 100% of the transient and about 30% of the steady-state inward currents and depolarizes the cell membrane by 5-12 mV. Second, inside-out membrane patches display a single-channel inward current induced by gluconate (40 mM) and blocked by ATP (200 microM) at the cytoplasmic side. The channels apparently show two sublevels (each of approximately 27-32 pS) with a total of 85-pS maximal conductance at -80 mV; the open probability follows a two-exponential mechanism. Thus, functional K(ATP) channels, composed of Kir6.1/SUR1, are present in frog Müller cells and contribute a significant part to the whole-cell K+ inward currents in the absence of ATP. Other inwardly rectifying channels, such as Kir4.1 or Kir2.1, may mediate the remaining currents. K(ATP) channels may help maintain glial cell functions during ATP deficiency.
幼体和成体两栖动物的视网膜和大脑能够在长时间缺氧状态下存活；这一发现表明存在功能性的K(ATP)通道。我们之前通过免疫细胞化学研究表明，成年青蛙视网膜中的神经胶质（穆勒）细胞表达K(ATP)通道及受体蛋白Kir6.1和SUR1，而视网膜神经元则表达Kir6.2和SUR2A/B（斯卡奇科夫等人，2001a：《神经报告》12：1437 - 1441；伊顿等人，即将发表：《神经报告》）。通过免疫细胞化学和电生理学方法，我们证明了成年青蛙和蝌蚪的穆勒细胞中存在Kir6.1/SUR1（K(ATP)）通道。在从成年青蛙和蝌蚪分离出的穆勒细胞中，使用有利于激活K(ATP)通道的条件（即不含ATP和精胺、含葡萄糖酸盐的细胞质透析溶液），我们得到了以下结果。首先，在全细胞记录中使用膜片钳技术，甲苯磺丁脲（一种K(ATP)通道阻滞剂）可阻断近100%的瞬态电流和约30%的稳态内向电流，并使细胞膜去极化5 - 12 mV。其次，外翻膜片在细胞质侧显示由葡萄糖酸盐（40 mM）诱导并被ATP（200 microM）阻断的单通道内向电流。这些通道在 - 80 mV时明显呈现两个亚水平（每个亚水平约为27 - 32 pS），最大电导总计85 pS；开放概率遵循双指数机制。因此，由Kir6.1/SUR1组成的功能性K(ATP)通道存在于青蛙穆勒细胞中，并且在无ATP的情况下对全细胞K+内向电流有显著贡献。其他内向整流通道，如Kir4.1或Kir2.1，可能介导其余电流。K(ATP)通道可能有助于在ATP缺乏时维持神经胶质细胞的功能。
