Near-membrane [Ca2+] transients resolved using the Ca2+ indicator FFP18.

Abstract

Ca2+-sensitive processes at cell membranes involved in contraction, secretion, and neurotransmitter release are activated in situ or in vitro by Ca2+ concentrations ([Ca2+]) 10-100 times higher than [Ca2+] METHODSSmooth muscle cells were enzymatically isolated from stomachs of the toad Bufo marinus according to described procedures (26). Measuring Spectral Properties. Excitation spectra of membrane-associated FFP18 at various [Ca2+] were recorded using a SPEX spectrofluorimeter. Emission was collected at 510 nm.FFP18 was applied to the external surface of cells by 10-to 30-min incubations in amphibian physiological saline (APS) containing 0.5-10 ,uM FFP18. Cell suspensions with different free [Ca2+] levels were produced by a described procedure (25). The concentration of membrane-associated FFP18 and the density of FFP18 molecules on the cell membrane were estimated using described methods (25).Abbreviations: SR, sarcoplasmic reticulum; [Ca+2]j, intracellular free Ca2+ concentration; APS, amphibian physiological saline. 5368The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Measurement of Intracellular Calcium

“…FFP18 and FIP18 are more recent indicators for measurement of near-membrane [Ca 2ϩ ] (75). The K d of FFP18 is three times greater than that of C 18 -fura 2, which enables more accurate measurements at higher [Ca 2ϩ ] (75,106,415). In addition, it is less lipophilic than C 18 -fura 2, resulting in faster diffusion out of the microinjection pipette into cells (106).…”

“…C 18 -fura 2 and FFP18 are relatively novel Ca 2ϩ indicators based on fura 2 (105,106), and FIP18 is based on indo 1. C 18 -fura 2 was synthesized by conjugating fura 2 to a lipophilic alkyl chain.…”

The Kinetics of Ca2+-Dependent Switching in a Calmodulin−IQ Domain Complex

“…The principal difference between the equilibrium and transient behaviors of the CaM-IQ domain complex is that the N-ter Ca 2+ -bound intermediate plays a significant role when Ca 2+ is rapidly increased to levels above 10 µM, as can occur locally during a neuronal Ca 2+ transient (33)(34)(35)(36)(37)(38). This intermediate does not form to a significant extent under equilibrium conditions, primarily because the C-ter Ca 2+ -binding sites, although slower kinetically, have an EC50(Ca 2+ ) of ~5 µM, while the value for the N-ter sites is ~ 35 µM (15).…”

Intracellular Calcium Ion Concentration Measurement Using a Phase-modulation Fluorescence Lifetime Method with Compensation for Phase Shift due to the Presence of Proteins

“…Ratiometric measurements are required for indicators that exhibit spectral shifts upon ion binding for quantitative ion measurements. Fura-2 7,8 and indo-1 11,12 can be used for this purpose, but cells are damaged by 340 to 380 nm ultraviolet light used for exciting the indicators, and the spectra of the indicators change due to interactions between the indicators and the protein in the cells. 13 The Cameleon GFP calcium sensor is also widely used for ratiometric measurements.…”

“…The measurement of intracellular free calcium ion concentrations ([Ca 2+ ]i) is widely used to examine the functions of cell components, such as ion channels and the effects of cell exposure to reagents. [1][2][3][4] [Ca 2+ ]i is often measured with a calcium indicator, called Cameleon, 5,6 which is a fusion protein of green fluorescent protein (GFP) and calmodulin, or a fluorescent calcium indicator of a low-weight molecule, such as fura-2 7,8 or calcium orange, 9 fura-red, 10 etc. In measurements using fluorescent calcium indicators of low molecular weight, the fluorescence intensity change of the calcium indicator determines [Ca 2+ ]i.…”