Hafting T, Sand O
Department of Biology, University of Oslo, Oslo, Norway.
Acta Physiol Scand. 2000 Oct;170(2):99-109. doi: 10.1046/j.1365-201x.2000.00766.x.
We have studied the activation of a high-conductance channel in clonal kidney cells from African green monkey (Vero cells) using patch-clamp recordings and microfluorometric (fura-2) measurements of cytosolic Ca2+. The single-channel conductance in excised patches is 170 pS in symmetrical 140 mM KCl. The channel is highly selective for K+ and activated by membrane depolarization and application of Ca2+ to the cytoplasmatic side of the patch. The channel is, thus, a large-conductance Ca2+-activated K+ channel (BK channel). Cell-attached recordings revealed that the channel is inactive in unstimulated cells. Extracellular application of less than 0.1 microM ATP transiently increased the cytosolic Ca2+ concentration ([Ca2+]i) to about 550 nM, and induced membrane hyperpolarization caused by Ca2+-activated K+ currents. ATP stimulation also activated BK channels in cell-attached patches at both the normal-resting potential and during membrane hyperpolarization. The increase in [Ca2+]i was owing to Ca2+ release from internal stores, suggesting that Vero cells express G-protein-coupled purinergic receptors (P2Y) mediating IP3-induced release of Ca2+. The P2Y receptors were sensitive to both uracil triphosphate (UTP) and adenosine diphosphate (ADP), and the rank of agonist potency was ATP >> UTP >/= ADP. This result indicates the presence of both P2Y1 and P2Y2 receptors or a receptor subtype with untypical agonist sensitivity. It has previously been shown that hypotonic challenge activates BK channels in both normal and clonal kidney cells. The subsequent loss of KCl may be an important factor in cellular volume regulation. Our results support the idea of an autocrine role of ATP in this process. A minute release of ATP induced by hypotonically evoked membrane stretch may activate the P2Y receptors, subsequently increasing [Ca2+]i and thus causing K+ efflux through BK channels.
我们利用膜片钳记录技术以及胞质Ca2+的微量荧光测定法（fura-2），研究了非洲绿猴肾克隆细胞（Vero细胞）中高电导通道的激活情况。在对称的140 mM KCl中，切除膜片上的单通道电导为170 pS。该通道对K+具有高度选择性，并可被膜去极化以及向膜片胞质侧施加Ca2+激活。因此，该通道是一种大电导Ca2+激活的K+通道（BK通道）。细胞贴附式记录显示，该通道在未受刺激的细胞中处于失活状态。胞外施加小于0.1 microM的ATP可使胞质Ca2+浓度（[Ca2+]i）短暂升高至约550 nM，并诱导由Ca2+激活的K+电流引起的膜超极化。ATP刺激还可在正常静息电位以及膜超极化期间激活细胞贴附膜片中的BK通道。[Ca2+]i的升高归因于内部储存库释放Ca2+，这表明Vero细胞表达介导IP3诱导的Ca2+释放的G蛋白偶联嘌呤能受体（P2Y）。P2Y受体对尿嘧啶三磷酸（UTP）和二磷酸腺苷（ADP）均敏感，激动剂效力顺序为ATP >> UTP >= ADP。这一结果表明存在P2Y1和P2Y2受体或具有非典型激动剂敏感性的受体亚型。先前已表明，低渗刺激可激活正常肾细胞和肾克隆细胞中的BK通道。随后KCl的丢失可能是细胞体积调节中的一个重要因素。我们的结果支持ATP在此过程中具有自分泌作用的观点。低渗诱发的膜拉伸诱导的微量ATP释放可能激活P2Y受体，随后增加[Ca2+]i，从而导致K+通过BK通道外流。
