CALCIUM TRANSIENTS IN STRIATED MUSCLE AND OTHER CELLS

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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.