Figure 9.

Effects of pH, pH gradients, and protein orientation on apparent compound potencies in SSME recordings and off-target compound effects: (A) Effects of 3 µM, 10 µM, and 30 µM DCPIB on TMEM175 currents activated using K⁺ concentration jumps employing different K⁺ concentrations. The non-activating solution contained 140 mM NaCl, the activating solution contained x = 2, 5, 10, or 50 mM KCl and (140 − x) mM NaCl. Currents were recorded using one 96-well sensor plate with the SURFE²R 96SE. Currents were normalized to the currents recorded with the respective K⁺ concentration in the absence of DCPIB. The bar plot shows average, normalized currents and SEM recorded from N = 8 sensors per condition; (B) pH dependence of IC₅₀ curves for 4-AP recorded using TMEM175 lysosomes with the SURFE²R N1. A solution exchange of 50 mM Na⁺ against 50 mM K⁺ was used to stimulate TMEM175. Datasets for each pH were measured on different sensors. Datasets were normalized to the peak current recorded in the absence of 4-AP before averaging. To indicate the pH dependence of absolute currents, we normalized each pH dataset a second time, using the average current obtained in the absence of 4-AP (I_max). Average peak currents and SEM, recorded from N = 5 sensors, are shown. I_max was fixed in the subsequent fitting with the equation I = I_max − (I_max − I_min)/(1 + (c/EC₅₀)ⁿ). The dataset recorded at pH 4.0 (orange) only consists of artifact currents that are independent of 4-AP concentration; (C) As in (B), but instead of pH values being equilibrated in both internal and external solutions, we applied a pH gradient as indicated, incubating with non-activating solution at pH 4.5 for 3 min to equilibrate the intra-lysosomal pH, before switching to non-activating solution at pH 7.6 defining the external pH ~700 ms in advance of the 50 mM K⁺ jump to stimulate TMEM175. Both datasets—symmetrical pH 7.6 (green) and pH gradient (blue) conditions—were recorded on the same sensor, with N = 5 sensors in total. Since no major impact of the pH conditions on I_max have been observed, averaged and normalized currents are shown (compare with Figure 4G); (D) Effect of pH gradients on compound potency recorded with the SURFE²R N1. The compounds are blinded, but the labels match with the labels shown in Figure 8. Experimental conditions as described in (C). Currents in the presence and the absence of a pH gradient were recorded with different sensors. Each sensor was used for four measurements in the following sequence: in the absence of compound, in the presence of 100 nM compound, in the presence of 10 µM compound, and in the absence of compound (wash-out). Datasets were normalized to the peak current recorded in the absence of compound before averaging. Average peak currents and SD from N = 3 sensors are shown; (E) Intra-lysosomal zinc application and its effects on TMEM175 recorded with the SURFE²R N1. Sensors were prepared with TMEM175 lysosomes that were pre-loaded via 1:10 dilution in zinc containing non-activating solution, followed by 10 pulses of sonication as described in the Methods section (Section 4.2.5). For each zinc concentration, we applied a solution exchange of 50 mM Na⁺ against 50 mM K⁺ in the absence of zinc, causing zinc to only be present inside the lysosomes. Average peak currents and SD from N = 5 sensors are shown, and no normalization was applied; (F) Measurements of compound effects on control lysosomes purified from HEK293 cells expressing endogenous TMEM175, recorded with the SURFE²R N1. A solution exchange of 50 mM Na⁺ against 50 mM K⁺ was used, both in the absence of the compound, and in the presence of the two highest compound concentrations used for EC₅₀ and IC₅₀ determination using the TMEM175 sample (Figure 6 and Figure 8A). The three different experimental conditions shown in one graph were applied on the same sensor. Average peak currents and SD from N = 4 sensors are shown, and no normalization was applied.