Abstract
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 β2-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 β2-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 β2-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.
Highlights
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
We aimed to address this hypothesis of higher local drug concentrations, quantifying concentrations of the fluorescent propranolol derivative BODIPY630/650-PEG8-S-propranolol (BY-propranolol)[10] immediately above the cell membrane and the bulk aqueous phase using fluorescence correlation spectroscopy (FCS)
Autocorrelation analysis of BY-propranolol fluorescence fluctuations detected in the FCS detection volume placed 3, 4 and 5 μm above the coverslip revealed concentrations of 5.1 ± 0.8 nM (n = 8), 3.2 ± 0.4 nM (n = 8) and 3.3 ± 0.5 nM (n = 8), respectively, all of which were higher than the 1.8 nM BY-propranolol concentration added (P < 0.05, t-test)
Summary
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, known that drugs are able to interact directly with the plasma membrane, potentially increasing local ligand concentrations around the receptor. The fundamental equations routinely used in the determination of pharmacological parameters, such as binding affinities, assume a freely diffusible and homogeneously distributed ligand in solution In experimental terms this means that the ligand concentration near the receptor is assumed to be the same as in the bulk aqueous phase. We use FCS to show that BY-propranolol concentrations in the immediate vicinity of the membranes of individual CHO cells are substantially higher than those in the bulk solution phase, and that these concentrations are influenced by the presence of the β2-adrenoceptor
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