Detection of changes in near-membrane Ca2+ concentration using a novel membrane-associated Ca2+ indicator.
Detection of changes in near-membrane Ca2+ concentration using a novel membrane-associated Ca2+ indicator.
复制标题
DOI：
10.1016/s0021-9258(17)37001-1
复制
发表时间：
1994-04
期刊：
影响因子：
--
通讯作者：
E. Etter;Michael A. Kuhn;Fredric S. Fay
中科院分区：
文献类型：
--
作者：
E. Etter;Michael A. Kuhn;Fredric S. Fay
一种钙离子指示剂已被合成和表征，其可用于监测细胞膜附近游离钙离子浓度（[Ca 2 +]）的快速变化。该指示剂被称为C18-Fura-2，由Fura-2分子与亲脂性烷基链结合组成，该亲脂性烷基链将插入细胞膜。当与低浓度的细胞膜缔合时，C18-Fura-2表现出具有大的斯托克斯位移和单个等吸光点的激发光谱，因此[Ca 2 +]可以比率计算。细胞相关C18-Fura-2的表观Ca 2+解离常数约为150 nM。C18-Fura-2在细胞膜中定向，使得荧光团面向其施加的一侧。使用C18-Fura-2记录细胞内[Ca 2 +]的快速变化，其响应于分离的平滑肌细胞中的膜去极化而发生。C18-Fura-2报告的[Ca 2 +]瞬变的初始上升比胞质Fura-2报告的[Ca 2 +]瞬变的上升快4至6倍。这一结果表明，C18-Fura-2位于质膜附近的网站的Ca 2+内流，并表明膜相关的Ca 2+指示剂可用于检测快速，局部的变化[Ca 2 +]，这是模糊的信号记录使用水溶性，散装胞质荧光Ca 2+指示剂。
A Ca2+ indicator has been synthesized and characterized which can be used to monitor rapid changes in the free Ca2+ concentration ([Ca2+]) immediately adjacent to cell membranes. This indicator, referred to as C18-Fura-2, consists of a Fura-2 molecule conjugated to a lipophilic alkyl chain which will insert into cell membranes. When associated with cell membranes in low concentrations, C18-Fura-2 exhibits an excitation spectrum with a large Stokes shift and a single isobestic point, thus [Ca2+] can be calculated ratiometrically. The apparent Ca2+ dissociation constant of cell-associated C18-Fura-2 is around 150 nM. C18-Fura-2 orients in the cell membrane so that the fluorophore is facing the side to which it was applied. C18-Fura-2 was used to record rapid changes in intracellular [Ca2+] which occurred in response to membrane depolarization in isolated smooth muscle cells. The initial rise of the [Ca2+] transient reported by C18-Fura-2 was four to six times faster than the rise of the [Ca2+] transient reported by cytosolic Fura-2. This result suggests that C18-Fura-2 was located at the plasma membrane near sites of Ca2+ influx and indicates that membrane-associated Ca2+ indicators can be used to detect rapid, localized changes in [Ca2+] which are obscured in signals recorded using water-soluble, bulk cytosolic fluorescent Ca2+ indicators.