Figure 3. Arachidonic Acid and synthetic chemicals activate TMEM175.

(A) Effects of Arachidonic Acid (ArA, 200 μM) on whole-LEL H⁺ currents in COS1 cells. The luminal solution contained NMDG-MSA with pH_L set to 4.6. (B) Summary of current density at −120 mV from isolated enlarged LELs as in (A) (n = 5 LELs, mean ± SEM, *** p <0.001). (C-D) Effects of ArA (100 μM) on whole-cell H⁺ currents in TMEM175-transfected HEK293T cells. Current density (−120 mV) before and after ArA application is summarized in (D) (n = 9 cells, mean ± SEM, ** p <0.01), A representative recording from a non-transfected control cell is shown in Supplemental Fig. S4A. (E-F) Effects of DCPIB (100 μM) on whole-LEL H⁺ currents in COS1 cells. Current density (−120 mV) is summarized in (F) (n = 10 LELs, mean ± SEM, *** p <0.001). (G-H) Effects of DCPIB (50 μM) on whole-cell H⁺ currents in TMEM175-transfected HEK293T cells. Current density (−120 mV) before and after DCPIB application is summarized in (H) (n = 8 cells, mean ± SEM, ** p <0.01). (I) Representative traces of whole-LEL H⁺ currents from WT HAP1 cells, TMEM175 KO cells, and KO cells re-expressing human TMEM175 (KO+hTMEM175) in response to ArA (100 μM) with pH_L = 4.6 and pH_C = 7.2. (J) Summary of whole-LEL H⁺ currents (−120 mV) from experiments as in (I) (WT/KO/KO+hTMEM175, n = 9/10/6, mean ± SEM, *** p <0.001). (K) Representative traces of whole-LEL H⁺ currents from WT HAP1 cells, TMEM175 KO cells, and KO cells re-expressing human TMEM175 (KO+hTMEM175) in response to DCPIB (100 μM) with pE_L = 4.6 and pH_C = 7.2. (L) Summary of whole-LEL H⁺ currents (−120 mV) from experiments as in (K) (WT/KO/KO+hTMEM175, n = 9/8/7, mean ± SEM, *** p <0.001).