Abstract
Drug-receptor interaction theory predicts that proportional receptor occupancy is a function of ligand concentration as defined by a ligand-receptor affinity constant, and is independent of receptor density. However, we previously observed that the EC50 of 5-HT reduced as the density of 5-HT3 receptors increased, suggesting an effect of receptor density on occupancy. The current study was designed to maximise variability in experimentally observed currents and confirm this apparent contradiction prospectively. Xenopus oocytes were injected with RNA encoding 5-HT3A receptors under conditions designed to achieve varying receptor expression levels and 5-HT-evoked currents measured using two electrode voltage clamp. Results from 99 oocytes showed that as the maximal peak current increased from 0.05 µA to 12.1 µA there was a 3.7-fold reduction in EC50. Since occupancy and conductance are directly related in this system, this indicates that for a given concentration of 5-HT, proportional occupancy increases with increased receptor density. We conclude that normalising data masks this correlation, and can result in reduced accuracy of pharmacological measurements. We propose a mechanistic explanation for our observations.
Highlights
Drug-receptor interaction theory predicts that proportional receptor occupancy is a function of ligand concentration as defined by a ligand-receptor affinity constant, and is independent of receptor density
The assumption that peak Imax is a good measure of receptor expression is logically well-founded since 5-HT3 receptors have a unitary conductance[4,5,6,7], and their desensitisation is minimal in the experimental system described here[3,8,9,10] (Fig. 1b, insert)
By using electrophysiology to quantify receptor expression, individual estimates of Imax and pEC50 are obtained from data collected under the same conditions
Summary
Drug-receptor interaction theory predicts that proportional receptor occupancy is a function of ligand concentration as defined by a ligand-receptor affinity constant, and is independent of receptor density. Since the magnitude of whole cell currents is dependent on channel expression levels, measured currents are frequently normalised to a specified (often maximally observed) value to eliminate substantial variance in response that arises because of these varying levels of channel expression. Such normalisation implies that Kd and EC50 are independent of channel density and eliminates the possibility of recognising covariance between maximal current and other pharmacological parameters that define the response. 5-HT3 receptors are ligand-gated ion channels that contain an integral ion channel and they are an ideal model for studying the relationships between agonist concentration, receptor expression and proportional occupancy, as RNA and DNA encoding them can readily be introduced into cells, and their expression on the cell-surface makes them amenable to electrophysiological measurements. When 5-HT binds at extracellular sites, it opens a transmembrane pore that allows ions to flow across the cell-surface membrane with
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