Imaging Ca(2+) entering the cytoplasm through a single opening of a plasma membrane cation channel.
Imaging Ca(2+) entering the cytoplasm through a single opening of a plasma membrane cation channel.
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DOI：
10.1085/jgp.114.4.575
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发表时间：
1999-10
期刊：
影响因子：
--
通讯作者：
Singer JJ
中科院分区：
文献类型：
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作者：
Zou H;Lifshitz LM;Tuft RA;Fogarty KE;Singer JJ
离散的局部荧光瞬态由于一个单一的质膜Ca 2+渗透阳离子通道的开口记录使用宽视场数字成像显微镜与fluo-3作为Ca 2+指示剂。这些瞬变，同时记录的单一通道电流使用全细胞电流记录配置的膜片钳技术。平滑肌细胞中的这种阳离子通道被咖啡因打开（格雷罗，A.，F.S.费伊和杰杰·辛格1994. 104：375-394）。局部荧光瞬变似乎发生在细胞膜上的随机位置，上升相的持续时间与通道开放的持续时间相匹配。此外，这些瞬变仅在存在足够的细胞外Ca 2+的情况下观察到，表明它们是由于来自浴溶液的Ca 2+流入。荧光瞬态的特征在于当通道打开时的初始快速上升阶段，随后是在长时间打开期间的较慢上升阶段。当通道关闭时，存在立即快速下降阶段，随后是较慢的下降阶段。计算机模拟的基础事件被用来解释的瞬变的时间过程。快速相主要是由于当通道打开或关闭时，通道附近的Ca 2+和Ca 2+结合的fluo-3梯度的建立或去除，而缓慢相是由于Ca 2+和Ca 2+结合的fluo-3扩散到细胞质中。由于短通道开口的瞬态有一个“Ca 2+火花样”的外观，这表明火花的上升和早期下降的组件（由于兰尼碱受体的开口）反映了荧光变化的快速阶段。这里提出的结果建议的方法来确定荧光瞬态和潜在的Ca 2+电流之间的关系，研究细胞内的本地化Ca 2+处理可能会发生从单一的Ca 2+通道开口，并本地化Ca 2+渗透性离子通道的质膜。
Discrete localized fluorescence transients due to openings of a single plasma membrane Ca2+ permeable cation channel were recorded using wide-field digital imaging microscopy with fluo-3 as the Ca2+ indicator. These transients were obtained while simultaneously recording the unitary channel currents using the whole-cell current-recording configuration of the patch-clamp technique. This cation channel in smooth muscle cells is opened by caffeine (Guerrero, A., F.S. Fay, and J.J. Singer. 1994. J. Gen. Physiol. 104:375–394). The localized fluorescence transients appeared to occur at random locations on the cell membrane, with the duration of the rising phase matching the duration of the channel opening. Moreover, these transients were only observed in the presence of sufficient extracellular Ca2+, suggesting that they are due to Ca2+ influx from the bathing solution. The fluorescence transient is characterized by an initial fast rising phase when the channel opens, followed by a slower rising phase during prolonged openings. When the channel closes there is an immediate fast falling phase followed by a slower falling phase. Computer simulations of the underlying events were used to interpret the time course of the transients. The rapid phases are mainly due to the establishment or removal of Ca2+ and Ca2+-bound fluo-3 gradients near the channel when the channel opens or closes, while the slow phases are due to the diffusion of Ca2+ and Ca2+-bound fluo-3 into the cytoplasm. Transients due to short channel openings have a “Ca2+ spark-like” appearance, suggesting that the rising and early falling components of sparks (due to openings of ryanodine receptors) reflect the fast phases of the fluorescence change. The results presented here suggest methods to determine the relationship between the fluorescence transient and the underlying Ca2+ current, to study intracellular localized Ca2+ handling as might occur from single Ca2+ channel openings, and to localize Ca2+ permeable ion channels on the plasma membrane.