CALCIUM TRANSIENTS IN STRIATED MUSCLE AND OTHER CELLS

摘要：钙离子(Ca~(2+))是最重要的离子之一。 生物学中的细胞内信使；为了能够理解 它的功能，必须能够测量细胞内的钙离子 活细胞中的浓度。现在有5种方法可供选择 为此：(1)钙选择微电极，(2)金属变色染料， (3)核磁共振；(4)荧光染料；(5)钙离子 -受调节的光蛋白。 这些方法中的每一种都有自己的一套优缺点； 没有一种方法对所有目的都是理想的。受钙离子调控的光蛋白有 特别适合用于肌肉，因为它们相对 对运动伪像免疫，并已用于大多数 心肌细胞内钙瞬变的研究。Aequorin，现在唯一的 光蛋白广泛存在，通常是从发光物质中分离出来的 水母Aequorea，但活性Aequorin最近由 基因克隆。然而，aequorin(天然的或重组的)有一个数字 缺点：(1)对钙离子快速变化的反应太慢 浓度，(2)它与镁和钙结合，结果是 对Ca2+的敏感性并不总是与特定应用的理想匹配。 这项研究的总体目标是构建更好的光蛋白。这个 第一步是研究钙离子敏感性、镁离子敏感性和 其他(稀有)天然光蛋白的动力学及其 同种异种。)我们已经知道有相当大的变化 在自然产生的光蛋白中的这些特性中。) 具有特别理想特性的光蛋白将被测序， 以及它们的载脂蛋白基因在可能的情况下通过 已建立的合作关系。预计有用的数量 因此，具有特别理想特性的稀有光蛋白可以 可用作钙离子指示剂。除此之外，它可能是 有可能进一步改善分子的某些特性，以及 通过定点突变更好地了解其功能。

ABSTRACT: The calcium ion (Ca2+) is one of the most important intracellular messengers in biology; in order to be able to understand its functions, one must be able to measure intracellular Ca2+ concentrations in living cells. There are now 5 methods available for doing this: (1) Ca-selective microelectrodes, (2) metallochromic dyes, (3) nuclear magnetic resonance, (4) fluorescent dyes, and (5) Ca2+ -regulated photoproteins. Each of these methods has its own set of advantages and disadvantages; none is ideal for all purposes. Ca2+ -regulated photoproteins are especially well suited for use in muscle because they are relatively immune to motion artifacts, and have been used for the great majority of studies of Ca2+ transients in heart muscle. Aequorin, now the only photoprotein widely available, is usually isolated from the luminescent jellyfish Aequorea, but active aequorin has recently been produced by gene cloning. However, aequorin (natural or recombinant) has a number of shortcomings: (1) it responds too slowly to rapid changes in Ca2+ concentration, (2) it binds Mg2+ as well as Ca2+ with the result that Ca2+ -sensitivity is not always ideally matched to specific applications. The overall goal of this research is to build a better photoprotein. The first step will be to study the Ca2+ -sensitivity, Mg2+ sensitivity, and kinetics of other (rare) naturally occurring photoproteins and their isospecies. (It is already known that there are considerable variations in these properties among naturally occurring photoproteins.) Photoproteins with particularly desirable properties will be sequenced, and the genes for their apoproteins cloned where possible through established collaborations. It is anticipated that useful quantities of rare photoproteins with particularly desirable properties can thus be made available for use as Ca2+ indicators. Beyond that, it may be possible to further improve certain characteristics of the molecule, and to better understand its function through site-directed mutagenesis.