Protein and acidosis alter calcium-binding and fluorescence spectra of the calcium indicator indo-1

Abstract

The fluorescent indicator indo-1 is widely used to monitor intracellular calcium concentration. However, quantitation is limited by uncertain effects of the intracellular environment on indicator properties. The goal of this study was to determine the effects of protein and acidosis on the fluorescence spectra and calcium dissociation constant (Kd) of indo-1. With 350 nm excitation light, the ratio of indo-1 fluorescence in the absence versus the presence of saturating Ca2+ at wavelength lambda (S lambda) and Kd increased with [protein]. At pH 7.3, Kd, S400, and S470, which were 210 nM, 0.033, and 1.433 in the absence of protein, increased to 808 nM, 0.161, and 2.641, respectively, by adding proteins from frog muscle and to 638 nM, 0.304, and 3.039, respectively, by adding proteins from rat heart. Effects of protein on indo-1 fluorescence were reduced at higher [indo-1]. Acidosis (pH 6.3) had separate effects, which were additive to those of protein: in the absence of protein, acidosis increased Kd to 640 nM; frog muscle proteins further increased Kd to 1700 nM. Acidosis also changed S lambda slightly. In summary, interaction with protein or protons alters indo-1 calcium-binding and fluorescence. These findings are consistent with several previous studies and suggest that indo-1 calibration constants need to be derived in the presence of appropriate types of protein, ratio of [indo-1]/[protein], and pH.

Resting cytoplasmic free Ca2+ concentration in frog skeletal muscle measured with fura-2 conjugated to high molecular weight dextran.

“…)They found that Rma~/R~in (a parameter similar to our R1/Ro) is several fold smaller in the mouse fibers than in salt solutions, but they also found that the indicator Ko in the fiber was verysimilar to that in the cell-free solutions. The latter finding appears to contradict the results from other laboratories, which have reported a substantial increase in the Ko of indo-1 by interaction with intracellular proteins (Hove-Madsen and Bers, 1992;Baker et al, 1994).…”

“…A number of studies on many Ca 2+ indicators have shown that the indicator properties are likely to be altered in the cytoplasm, probably by binding to cellular macromolecules: fura-2 (Konishi et al, 1988;Uto, Arai, andOgawa, 1991), indo-1 (Hove-Madsen andBers, 1992;Westerblad and Allen, 1993;Baker et al, 1994), fluo-3 , fura red , and aequorin (Blatter and Blinks, 1991). The present results suggest that fura-2 conjugated to dextran can still bind to cellular proteins.…”

“…On the other hand, another very well controlled study, which used both Ca2+-sensitive microelectrodes and aequorin, reports that resting [Ca2+]i is below the detection limit of their methods, and gives [Ca2+]i values of 0.04-0.06 oLM as an upper limit. Major problems related to the use of these indicators appear to be the binding of the indicators to intracellular constituents, which alters Ca ~+ affinity, the optical signals of the indicators, or both (e.g., Konishi, Olson, Hollingworth, and Baylor, 1988;Blatter and Blinks, 1991;Baker, Brandes, Schreur, Camacho, and Weiner, 1994).…”

Measurement of Intracellular Calcium

“…In addition, intracellular environments also affect spectral properties of some Ca 2ϩ indicators. It has been demonstrated that indo 1 and fura 2 exhibit different emission/ absorption spectra in intracellular environments compared with buffers (19,161,288). The discrepancy may be caused in part by the absorption/scattering by intracellular constituents (288) and changes in the polarity (288,289) and/or the viscosity (111, 362) of the environment.…”

Epidermal keratinocyte polarity and motility require Ca2+ influx through TRPV1

“…The 10% value was chosen based on earlier reports of the Indo-1 ratio in cells and the relative intensity changes measured by other fluorescent Ca 2+ indicators in migrating fish keratinocytes (Baker et al, 1994;Lee et al, 1999;Doyle et al, 2004;Huang et al, 2009). This value provided an objective criterion for analysis and enabled good separation between the background variation of the lamellipodial intensity and the Ca 2+ sparks.…”