Micro-pharmacokinetics: Quantifying local drug concentration at live cell membranes

Fundamental equations for determining pharmacological parameters, such as the binding affinity of a ligand for its target receptor, assume a homogeneous distribution of ligand, with concentrations in the immediate vicinity of the receptor being the same as those in the bulk aqueous phase. It is, however, known that drugs are able to interact directly with the plasma membrane, potentially increasing local ligand concentrations around the receptor. We have previously reported an influence of ligand-phospholipid interactions on ligand binding kinetics at the β(2)-adrenoceptor, which resulted in distinct “micro-pharmacokinetic” ligand profiles. Here, we directly quantified the local concentration of BODIPY630/650-PEG8-S-propranolol (BY-propranolol), a fluorescent derivative of the classical β-blocker propranolol, at various distances above membranes of single living cells using fluorescence correlation spectroscopy. We show for the first time a significantly increased ligand concentration immediately adjacent to the cell membrane compared to the bulk aqueous phase. We further show a clear role of both the cell membrane and the β(2)-adrenoceptor in determining high local BY-propranolol concentrations at the cell surface. These data suggest that the true binding affinity of BY-propranolol for the β(2)-adrenoceptor is likely far lower than previously reported and highlights the critical importance of understanding the “micro-pharmacokinetic” profiles of ligands for membrane-associated proteins.