Diazepam is not a direct allosteric modulator of α 1 -adrenoceptors, but modulates receptor signaling by inhibiting phosphodiesterase-4.

α _1A - and α _1B -adrenoceptors (ARs) are G protein-coupled receptors (GPCRs) that are activated by adrenaline and noradrenaline to modulate smooth muscle contraction in the periphery, and neuronal outputs in the central nervous system (CNS). α _1A - and α _1B -AR are clinically targeted with antagonists for hypertension and benign prostatic hyperplasia and are emerging CNS targets for treating neurodegenerative diseases. The benzodiazepines midazolam, diazepam, and lorazepam are proposed to be positive allosteric modulators (PAMs) of α ₁ -ARs. Here, using thermostabilized, purified, α _1A - and α _1B -ARs, we sought to identify the benzodiazepine binding site and modulatory mechanism to inform the design of selective PAMs. However, using a combination of biophysical approaches no evidence was found for direct binding of several benzodiazepines to purified, stabilized α _1A - and α _1B -ARs. Similarly, in cell-based assays expressing unmodified α _1A - and α _1B -ARs, benzodiazepine treatment had no effect on fluorescent ligand binding, agonist-stimulated Ca ²⁺ release, or G protein activation. In contrast, several benzodiazepines positively modulated phenylephrine stimulation of a cAMP response element pathway by α _1A - and α _1B -ARs; however, this was shown to be caused by off-target inhibition of phosphodiesterases, known targets of diazepam. This study highlights how purified, stabilized GPCRs are useful for validating allosteric ligand binding and that care needs to be taken before assigning new targets to benzodiazepines.