The development of Ca2+ indicators: a breakthrough in pharmacological researchThe ‘pre-quin2 era’The appearance of quin2New indicators, growing successFrom chemical indicators to gene productsAcknowledgementsReferences|Research FocusThe development of Ca21indicators: a breakthrough inpharmacological researchJacopo MeldolesiDIBIT, Department of Neuroscience and Center of Excellence in Physiopathology of Cell Differentiation, University Vita-Salute SanRaffaele and San Raffaele Scientific Institute, via Olgettina 58, Milano, ItalyThe development, beginning in 1979, of fluorescentCa21-specific indicators as research tools has revolutio-nized transmembrane signaling studies. In this article,the state of the art in the ‘pre-Ca21-indicator’ era andthe rationale for the development of indicators trappedin the cytosol to investigate the cytosolic concentrationof Ca21in mammalian cells are summarized. Sub-sequent extension of these studies to the level of thesingle cell, together with the unique impact that Ca21indicators have had on signaling research and the intro-duction of specific, fluorescent gene constructs thatprovide direct, high-resolution information about theintracellular concentration of Ca21, are also discussed.Beginning in 1979, a new class of experimental tools wasdeveloped that has revolutionized transmembrane signal-ing, one of the main fields of basic pharmacology. Thediscovery of these cytosolic Ca2þindicators did not occur byserendipity. Instead, this discovery was a preciselyplanned and elegantly executed one-man enterprise, theman being Roger Y. Tsien, a young scientist who wasstudying for a PhD in Physiology and had an extraordinary‘touch’ in organic chemistry. In this article, I illustrate thisenterprise, starting with the state of the art at the time thework began.The ‘pre-quin2 era’In the 1970s the role of Ca2þas the most important secondmessenger was already generally accepted. Signalingevents were known to be triggered and controlled bychanges of the cytosolic concentration of free Ca2þ{[Ca2þ]c}, in dynamic equilibrium not only with theextracellular [Ca2þ] but also with intracellular Ca2þstores. Knowledge about [Ca2þ]cwas, however, confusedand in many cases openly contradictory. This confusionarose because none of the procedures employed was fullysatisfactory, and the cross-checking of the results obtainedusing different procedures was not common.Let us consider some of the procedures that wereavailable at the time. Subcellular fractionation, which hadbeen instrumental in the study of protein and lipiddistribution, provided misleading results with respect toCa2þbecause of the uncontrolled release and uptakeevents that occurred after cell homogenization. Analysis of45Ca fluxes into and from living cells is still employed,however, to investigate the dynamics of intracellularstores rather than the cytosolic pool. The study of otherions, such as86Rb, flowing through Ca2þ-dependentchannels was risky because it depends not only on[Ca2þ]cbut also on a variety of other processes. Directassays were problematic on two levels: the introduction ofthe recording tools into the cells and the analysis of thesignals. Therefore, as pointed out by Tsien shortly after hisdiscovery [1] during this era, the study of [Ca2þ]cwas‘limited to robust and well anchored, large cells that cantolerate insertion of ion-selection electrodes or microinjec-tion of Ca2þindicators or buffers into one cell at a time’(e.g. muscle fibers, squid giant axons and limulusphotoreceptors). Smaller cells could also be studied,although only following traumatic treatments, such asfusion with erythrocyte ghosts or liposomes filled with theprobe, hypo-osmotic shock or scraping. We now know thatthe consequences of these treatments on Ca2þhomeostasiswere often devastating [2].With respect to the probes, let us consider two types.(i) The photoprotein aequorin, at the time employed in onlya few highly specialized laboratories, was resuscitatedsuccessfully in the 1990s, when gene-expression tech-niques enabled it to be used to study specific subcellularstructures. (ii) Two azo dyes, arsenazo III and antipyrilazoIII, were characterized by serious drawbacks such as lowCa2þand Mg2þselectivity, low affinity for Ca2þandcomplex Ca2þbinding stoichiometry [2].In 1979, Tsien was working in the Department ofPhysiology at the University of Cambridge (UK). From hisprevious experience with Ca2þ-selective microelectrodes[3–5] and fluorescent probes, used to investigate mem-brane potential [6], Tsien realized that new indicatorswere needed to monitor the kinetics of [Ca2þ]cchanges.The properties required for these indicators were to affectcellular Ca2þhomeostasis only moderately and in apredictable way, generating at the same time easilydetectable fluorescent signals that changed appropriatelyfollowing binding of the cation. The great idea, worked onin 1979 and published in April 1980 [7], started from thebest-known Ca2þchelator EGTA, which, however, cannotbe used as an indicator because it absorbs light in the farultraviolet range and is not fluorescent. The result was thesynthesis of ‘rationally designed, high-affinity buffers andoptical indicators for Ca2þin which methylene linksbetween oxygen and nitrogen of EGTA are replaced bybenzene rings’ [7]. The molecule that was better charac-terized in the initial studies, BAPTA [1,2-bis (2-amino-phenoxy) ethane-N,N,N0,N0-tetraacetic acid] (Figure 1),Corresponding author: Jacopo Meldolesi ([email protected]).Update TRENDS in Pharmacological Sciences Vol.25 No.4 April 2004www.sciencedirect.comalthough poor as a [Ca2þ]cindicator, is very popular inlaboratories throughout the world as the ideal intracellu-lar Ca2þbuffer. This is because, compared with EGTA,BAPTA is less affected by pH changes, has a higher Ca2þand Mg2þselectivity and is much faster at binding andreleasing Ca2þ.The appearance of quin2In the article published in 1980 by Tsien [7], severalanalogs, distinct in terms of their Ca2þaffinity and Ca2þand Mg2þselectivity, were described in addition to BAPTA.These molecules were already matching, at least inperspective, many of the dreams of scientists studyingCa2þ, although with a big limitation. Similar to the azodyes that were already available, these compounds weremembrane impermeant. This was a problem that Tsiensolved in a surprisingly short time. In April 1981, hedemonstrated that Ca2þchelators, when