Quantitative measurement of cAMP concentration using an exchange protein directly activated by a cAMP-based FRET-sensor.

Abstract:

:Förster resonance energy transfer (FRET)-based biosensors for the quantitative analysis of intracellular signaling, including sensors for monitoring cyclic adenosine monophosphate (cAMP), are of increasing interest. The measurement of the donor/acceptor emission ratio in tandem biosensors excited at the donor excitation wavelength is a commonly used technique. A general problem, however, is that this ratio varies not only with the changes in cAMP concentration but also with the changes of the ionic environment or other factors affecting the folding probability of the fluorophores. Here, we use a spectral FRET analysis on the basis of two excitation wavelengths to obtain a reliable measure of the absolute cAMP concentrations with high temporal and spatial resolution by using an "exchange protein directly activated by cAMP". In this approach, FRET analysis is simplified and does not require additional calibration routines. The change in FRET efficiency (E) of the biosensor caused by [cAMP] changes was determined as DeltaE = 15%, whereas E varies between 35% at low and 20% at high [cAMP], allowing quantitative measurement of cAMP concentration in the range from 150 nM to 15 microM. The method described is also suitable for other FRET-based biosensors with a 1:1 donor/acceptor stoichiometry. As a proof of principle, we measured the specially resolved cAMP concentration within living cells and determined the dynamic changes of cAMP levels after stimulation of the Gs-coupled serotonin receptor subtype 7 (5-HT7).

journal_name

Biophys J

journal_title

Biophysical journal

authors

Salonikidis PS,Zeug A,Kobe F,Ponimaskin E,Richter DW

doi

10.1529/biophysj.107.125666

subject

Has Abstract

pub_date

2008-12-01 00:00:00

pages

5412-23

issue

11

eissn

0006-3495

issn

1542-0086

pii

S0006-3495(08)78964-1

journal_volume

95

pub_type

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