Figure 7.

Effect of Histidine to Alanine mutations on allosteric modulation of protons by metal ions (a–g) and ogerin (h) and lorazepam (i). Functional assays were conducted with HEK293T cells transiently transfected with the receptor and GloSensor cAMP reporter. Concentration-responses were determined under a specific pH condition close to the proton potency at a corresponding receptor, specifically, pH 6.80 for WT, H17A, and H245A; pH 6.10 for H20A; pH 6.60 for H84A and H175A; and pH 6.40 for H89A, H159A, H169A, and H269A. Results were normalized to corresponding basal (1.0, in the absence of modulators) and represented mean (fold of basal) ± SEM from a minimum of 3 assays, each in a quadruplicate set. Curves were analyzed in Prism 8 using the built-in four-parameter logistic or Bell-shaped function. Pharmacological parameters were listed in Table S1.