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. Author manuscript; available in PMC: 2024 Jul 20.
Published in final edited form as: Mol Cell. 2023 Jun 29;83(14):2524–2539.e7. doi: 10.1016/j.molcel.2023.06.004

Figure 5. LAMP binding antagonizes DCPIB activation of TMEM175.

Figure 5.

(A) Sample I-V curves of Cs+ currents from TMEM175 at various DCPIB concentrations. TMEM175 was expressed in HEK293 cells alone or with LAMP-1, LAMP-2, or LAMP-1 deletion mutant. Outward Cs+ currents were recorded using whole-cell patch clamp at pH 7.4.

(B) Concentration-dependent DCPIB activation of TMEM175 or TMEM175 in complex with various LAMP protein constructs at 100 mV. Data for DCPIB activation of TMEM175 and TMEM175/LAMP-1-TM complex were normalized against their respective currents at 100 μM DCPIB and were fitted to the Hill equation with EC50=3.6±0.3 μM, n=1.4±0.1 for TMEM175 and EC50=3.5±0.2 μM, n=1.2±0.1 for TMEM175/LAMP-1-TM complex. With markedly reduced currents and lower DCPIB efficacy of TMEM175 upon LAMP inhibition, all data from TMEM175 in complex with LAMP-1, LAMP-2, or LAMP-1-ΔdLAMP were normalized against the averaged TMEM175 currents at 100μM DCPIB. Data are represented as mean ± SEM (n=10 independent experiments).

(C) Cs+ current density from TMEM175 and TMEM175 in complex with various LAMP constructs measured at 100 mV in whole-cell recordings with or without 10 μM DCPIB. Data are represented as mean ± SEM (n=10, *** p <0.001, ** p <0.01).

(D) Sample I-V curves and proton current density (at −100 mV) from TMEM175 and TMEM175 in complex with various LAMP constructs recorded with or without 10 μM DCPIB. The proton currents were recorded with pH 7.4 (as background control) or 5.5 in the bath and pH 7.4 in the pipette. NMDG+ was used as monovalent in both bath and pipette solutions. Data are represented as mean ± SEM (n=10, *** p <0.001, ** p <0.01).

(E) Sample I-V curves and Cs+ current density (at 100 mV) from TMEM175(T395W) mutant with or without LAMP-1 or 2 co-expression. The Cs+ currents were recorded at pH 7.4 with or without 10 μM DCPIB in the bath (extracellular). Data are represented as mean ± SEM (n=10, ** p <0.01).

(F) Sample I-V curves and proton current density (at −100 mV) from TMEM175(T395W) mutant with or without LAMP-1 or 2 co-expression. The proton currents were recorded with pH 5.5 with or without 10 μM DCPIB in the bath. NMDG+ was used as monovalent in both bath and pipette solutions. Data are represented as mean ± SEM (n=10, ** p <0.01).