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alcium entry through store-operated and other Ca²⁺ permeable channels is an electrogenic process and thus is driven by a combination of chemical (concentration gradient) and electrical (membrane potential) forces¹. Is it therefore reasonable and prudent to screen for modulators of calcium entry using fluorescent calcium reporter dyes yet totally ignore any effects modulators may have on membrane potential? Transmembrane depolarization does in fact reduce Ca²⁺ entry into HL-60 cells, to cite one example². Applied to High Throughput Screening (HTS), if for example an acutely toxic compound depolarized the membrane potential could the predicted decrease in Ca²⁺ entry be misidentified as receptor antagonism instead of cytotoxicity?

A simple yet impractical solution in HTS is to screen compounds twice, once using Fluo-4 AM dye loaded cells for measuring calcium changes and again with cells loaded using membrane potential dye. By contrast there is a more practical solution described as ouble-parameter fluorescent measurementsup>3 where cells are concomitantly dye loaded using the UV calcium reporter dye Indo-1 AM and membrane potential dye DisBac₂(3). In this study, using a fluorescence spectrophotometer, Ca²⁺ entry and chloride-dependent depolarization were observed in the same cells.

The author modified this technique using the HTS platform Hamamatsu FDSS-6000. The ratiometric dye Fura-2 AM is used yet only excited using 380 nm and not 340 nm. At excitation 380 nm emission 540 nm calcium binding to Fura-2 results in a decrease in fluorescence. The Molecular Devices No Wash Membrane Kit (MP Dye) is also used, with excitation at 480 nm and emission at 540 nm. Excitation is sequential not simultaneous, with a cycle time of about 1.5 sec. In this model Ca²⁺ entry results in a decrease in fluorescence (Fura-2), while transmembrane potential (measured using MP Dye) can hyperpolarize or depolarize. Such an example is shown in Figure 1 where both compounds induced calcium entry, as expected. Interestingly, membrane potential changes were different, with one compound inducing hyperpolarization, the other depolarization.

Figure 1. Cells dye loaded using both Fura-2 AM and Membrane Potential dyes. Compound (Cmpd) 1 or 2 (30 final each) added at the arrow and data collected in 1.5 sec intervals. Both compounds induced Ca²⁺ entry but either depolarized or hyperpolarized transmembrane potential. (n=6 per group, SEM is shown).

Apropos technical considerations the quencher in the MP Dye quenches Fura-2 AM extracellular signal, so MP Dye is added without washing off extracellular Fura-2 AM. The hardware configurations and software protocols on the FDSS-6000 are readily available. Assay run time is not increased as both calcium and membrane potential changes are collected in the same experiment, a true multiplexing format.In summary it is the author suggestion that for HTS campaigns measuring Ca²⁺ entry collecting both calcium and membrane potential measurements is now practical and relevant, based on results heretofore only available using spectrofluorometers ^4,5.

References
1. Bird G. St.J. and Putney J.W. Jr.: Fluorescent Indicators-Facts and Artifacts. In Putney J.W. Jr. (ed.) Calcium Signaling 2nd Edition. Boca Raton FL: CRC Press, 2006:76.

2. Pittet D, Di Virgilio F, Pozzani T, Monod A, Lew DP: Correlation between Plasma Membrane Potential and Second Messenger Generation in the Promyelocyte Cell Line HL-60. J. Biol. Chem. 1990; 265: 14256-14263.

3. Kremer SG, Zeng W, Skorecki KL: Simultaneous fluorescence measurement of calcium and membrane potential responses to endothelin. Am. J. Physiol. 1992; 263: C1302-C1309.

4. Montero, M. J. Garcia-Sancho, J. Alvarez. Activation by Chemotactic Peptide of a Receptor-operated Ca²⁺ Entry Pathway in Differentiated HL60 cells. J. Biol. Chem. 1994; 269: 29451-29456.

5. Laskey, R.E., D.J. Adams, M. Cannell, C. van Breeman. Calcium entry-dependent oscillations of cytoplasmic calcium concentration in cultured endothelial cell monolayers. Proc. Natl. Acad. Sci. 1992; 89:1690-1694.

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