Fluorescence correlation spectroscopy is a noninvasive technique for studying ligand-receptor interactions in live cells as shown for β2-adrenergic receptors

Fluorescence correlation spectroscopy (FCS) is a noninvasive technique for high-resolution spatial and temporal analysis of extremely low concentrated biomolecules. FCS is based on measuring fluctuations in fluorescence intensity caused by single fluorescently labelled molecules diffusing through a tiny confocal volume element, which is generated by a focused laser beam with a defined detection volume size of about 1 fL. The photons emitted by the illuminated molecules within this focus are recorded in a time-resolved manner via single-photon detection avalanche photo-diodes. The recorded fluorescence fluctuations can be statistically evaluated using autocorrelation analysis obtaining local concentrations, diffusion coefficients, and characteristic rate constants of molecular interactions. The time required for the passage of fluorescent molecules through the volume element is determined by the diffusion coefficient, which is related to the size and shape of a molecule. FCS provides the ability to distinguish between molecules based on the speed of their diffusion and therefore, enables simultaneous detection of both fast and slow moving molecules. For ligand receptor interactions, the diffusion coefficient of a free fluorescent ligand decreases significantly after binding to a much slower diffusing membrane associated receptor. Therefore, FCS is a powerful biophysical tool for studying ligand-receptor binding on the molecular level in their native environment on living cells without the need for separating unbound from bound ligand.

In order to investigate the binding behaviour of β₂-adrenergic receptors, noradrenaline was labelled with the fluorescent dye Alexa Fluor 532 (Alexa-NA). The relative specificity for the β₂AR as well as the agonistic profile of this ligand has been shown in former publications [1, 2]. After incubation of alveolar type II cells (A549) with 5 nM Alexa-NA, the focus was positioned to the upper plasma membrane by motor-aided scanning of the cell in the z-direction. At the position of the half-maximal fluorescence of the upper membrane, the focus took in fast diffusing free Alexa-NA molecules and slow diffusing ligand bound to the receptor as well. Additionally, the lower part of the focus was localized in the cytoplasm below the plasma membrane, allowing the detection of internalized receptor-ligand complex. After evaluation of the autocorrelation curve, three different diffusion time constants could be discriminated [3]. Freely diffusing Alexa-NA molecules showed a diffusion time constant τ_free of 0.06±0.004ms. Further, an averaged fast diffusion time constant τ_bound1 of 1.4±1.1ms and a slow diffusion time constant τ_bound2 of 34.7±14.1ms was found for receptor-ligand complexes in the plasma membrane with unrestricted and hindered lateral mobility, respectively. Quantitative analysis of the autocorrelation curves revealed a total binding of 33±6.8% from a given concentration of 5 nM Alexa-NA, distributed to 1.21±0.12 nM for τ_bound1 and 0.43±0.22 nM for τ_bound2. Thus, influences of pharmacological active compounds on the binding behaviour of β₂-adrenergic receptors on living cells can be monitored on the molecular level in real time.

References: [1] Hegener O, Prenner L, Runkel F et al. Biochemistry 2004; 43: 6190–6199

[2] Prenner L, Sieben A, Zeller K et al. Biochemistry 2007; 46: 5106–5113

[3] Sieben A, Prenner L, Sorkalla T et al. Biochemistry 2009; 48: 3477–3482