Measuring drug concentrations where it matters: the influence of subcellular ligand distribution on cellular pharmacology

When the pharmacology of new drugs is assessed it is assumed that the concentration of drug that the receptor is exposed to is the same as that in bulk aqueous phase. Lipophilic and/or basic drugs can, however, interact directly with the membrane surrounding the receptor, potentially concentrating drug close to the receptor. This rotation project will use cutting-edge biophysical imaging techniques to quantify the local drug concentrations of two fluorescent adenosine A1 receptor (A1-AR) ligands with different physicochemical properties to assess whether non-specific membrane interactions influence observed receptor pharmacology.

Fluorescence correlation spectroscopy (FCS) is a microscopy technique that measures fluctuations in fluorescence intensity from a small (~0.2fL) defined confocal volume, allowing both diffusion time and concentration to be determined for a fluorescent compound. The FCS detection volume will be positioned at various heights above the plasma membrane of cells expressing the A1-AR to measure the concentration of drug close to the receptor and in bulk aqueous phase. These concentrations will be compared to existing pharmacological data to determine whether differences observed can be related to their different physicochemical properties and subsequent concentration in the local vicinity of the receptor.