pH regulates potassium conductance and drives a constitutive proton current in human TMEM175

Abstract

Human TMEM175, a noncanonical potassium (K⁺) channel in endolysosomes, contributes to their pH stability and is implicated in the pathogenesis of Parkinson's disease (PD). Structurally, the TMEM175 family exhibits an architecture distinct from canonical potassium channels, as it lacks the typical TVGYG selectivity filter. Here, we show that human TMEM175 not only exhibits pH-dependent structural changes that reduce K⁺ permeation at acidic pH but also displays proton permeation. TMEM175 constitutively conducts K⁺ at pH 7.4 but displays reduced K⁺ permeation at lower pH. In contrast, proton current through TMEM175 increases with decreasing pH because of the increased proton gradient. Molecular dynamics simulation, structure-based mutagenesis, and electrophysiological analysis suggest that K⁺ ions and protons share the same permeation pathway. The M393T variant of human TMEM175 associated with PD shows reduced function in both K⁺ and proton permeation. Together, our structural and electrophysiological analysis reveals a mechanism of TMEM175 regulation by pH.

Fig. 1.. K⁺ conductivity of human TMEM175 regulated by pH.

(A) Representative current-voltage (I-V) curves obtained in whole-cell configuration from HEK293T cells expressing hsTMEM175 WT at pH 7.4 and 5.5. (B) Averaged currents measured at either 100 or −100 mV from (A) for both hsTMEM175 WT at pH 7.4 and 5.5 (N = 5), showing the difference between pH 7.4 and pH 4.5. *P < 0.05. (C) Representative I-V curves obtained in whole-cell configuration from HEK293T cells expressing hsTMEM175 WT at pH 7.4 and pH 4.5. (D) Averaged currents measured at 100 mV from (C) for both hsTMEM175 WT at pH 7.4 and pH 4.5 (N = 5), showing the difference between pH 7.4 and pH 4.5. *P < 0.05. (E) Representative I-V curves obtained in whole-cell configuration from HEK293T cells expressing hsTMEM175 Q65P mutant at pH 7.4 and pH 4.5. (F) Averaged currents at 100 mV of hsTMEM175 Q65P at pH 7.4 and pH 4.5 (N = 5), showing the difference at pH 7.4 or pH 4.5. *P < 0.05.

Fig. 2.. Proton conductance by TMEM175 WT and PD-associated mutants.

(A) Representative I-V curves obtained in whole-cell configuration from HEK293T cells without expressing hsTMEM175 at pH 7.4 and pH 4.5. The pipette solution contains 150 mM Cs⁺, and 150 mM Na⁺ was used in the bath solution. (B) Representative I-V curves from HEK293T cells expressing WT hsTMEM175 at pH 7.4 and pH 4.5, with 150 mM NMDG⁺ in the bath solution. (C) Representative I-V curves obtained in whole-cell configuration from HEK293T cells expressing hsTMEM175 M393T mutant at pH 7.4 and pH 4.5. (D) Averaged currents at 100 and −100 mV of control, hsTMEM175 WT, Q65P, and M393T at pH 7.4 and pH 4.5 (N = 5), showing the difference at pH 7.4 or pH 4.5. *P < 0.05; **P < 0.01.

Fig. 3.. Proton conductance and selectivity of human TMEM175.

(A) Representative I-V curves of hsTMEM175 WT at various pH as indicated showing the drift of the currents at −100 and +100 mV. (B) Averaged currents at −100 mV of hsTMEM175 WT from (A) as a function of pH values. Data are presented as means ± SEM for n = 4 (pH 5.5) and n = 3 (other pH). (C) E_rev as a function of pH values from experiments in (A). Data are presented as means ± SEM for n = 4 (pH 5.5) and n = 3 (other pH). (D) Conductance at −100 mV as a function of pH values derived from (B) and (C). (E) Representative I-V curves of hsTMEM175 WT at pH 7.4 and pH 4.5, showing the ion selectivity for proton over potassium, sodium, cesium, and NMDG⁺ ions.

Fig. 4.. The assembly mechanism of human TMEM175.

(A) 3D cryo-EM density of human TMEM175. Dimensions of the TMEM175 dimer are ~60 × 60 × 60 Å. (B) Ribbon diagrams of the human TMEM175 structure with the subunits colored in cyan and wheat, respectively. The 12 helices in one of the two subunits are labeled by numbers in magenta. (C) Sequence alignment of TM1 and TM7 across species and structural comparison of human TMEM175 NTD and CTD. In the aligned sequences, the conserved RxxxFSD motif is highlighted in gray, and the conserved gate residue is highlighted in red. Residues involved in K⁺ coordination are colored in blue, yellow, and red for hydrophilic layers 3, 2, and 1, respectively.

Fig. 5.. Ion permeation pore of human TMEM175.

(A) Cross section through the pore showing the surface electrostatic potential of the pore and the pore entrances at the luminal and cytosolic sides (pH 6.8 structure model). (B) The surface electrostatic potential of the luminal face of the channel. (C) The ion permeation pore of hsTMEM175 at pH 7.4 formed by TM1 and TM7 with restriction residues in sticks representing an open conformation. (D) The pore radii of pH 5.5 (blue), pH 6.8 (green), and pH 7.4 (red) hsTMEM175 structures along the pore axis calculated using the HOLE program (44). The conserved gate residues are labeled in red, and residues coordinating K⁺ are colored in green. (E) K⁺ sites coordinated by hydrophilic residues lining the ion permeation pore (pH 7.4). The conserved gate residues are labeled in red, and residues coordinating the three K⁺ ions are labeled in green. Cryo-EM density for the K⁺ ions are contoured at 2.1 σ and shown in gray. (F) The ion permeation pore of hsTMEM175 at pH 5.5 formed by TM1 and TM7 with restriction residues in sticks, representing a closed conformation for K⁺ permeation. (G) One K⁺ site coordinated by hydrophilic residues lining the ion permeation pore (pH 5.5). The conserved gate residues are labeled in red, and residues coordinating the K⁺ site are labeled in green. Cryo-EM density for the K⁺ ions are contoured at 1.3 σ and shown in gray.

Fig. 6.. Proton permeation pathway of human TMEM175.

(A) Selected water distribution in and around the hsTMEM175 structure from the molecular dynamics simulation. TM1 and TM7 of hsTMEM175 are shown in a ribbon diagram, water molecules are displayed as red spheres, and the ion permeation pore is indicated by an arrow. (B) Water molecules in the ion permeation pore of TMEM175. (C) Representative I-V curves of hsTMEM175 WT at conditions as indicated, showing the inhibition of TMEM175 conductivity by Zn²⁺ and 4-AP. (D) Averaged currents at −100 mV of hsTMEM175 WT from (C) at pH 4.5 in the absence and presence of 100 μM Zn²⁺ or 100 μM 4-AP. Data are presented as means ± SEM for n = 5.

Fig. 7.. Residues critical for potassium ion and proton permeation.

(A) Pore-lining residues in hsTMEM175 that we tested in this study for functional involvement in proton and K⁺ conductance. (B) Representative I-V curves of hsTMEM175 mutant I271W at pH 7.4 and pH 4.5, showing the abolishment of K⁺ and proton currents. (C) Representative I-V curves of hsTMEM175 mutants S45A at pH 7.4 and pH 4.5, showing abolished K⁺ conductance but normal proton conductance. (D) Representative I-V curves of hsTMEM175 mutants T49V at pH 7.4 and pH 4.5, showing abolished K⁺ conductance and reduced proton conductance. (E) Representative I-V curves of hsTMEM175 mutant T274A at pH 7.4 and pH 4.5, showing the abolishment of both proton and K⁺ currents. (F and G) Representative I-V curves of hsTMEM175 mutants D279A, D279N, D283A, and D283N at pH 7.4 and pH 4.5, showing their effects on K⁺ and proton conductance. (H) Averaged currents at 100 mV of pH 7.4 or −100 mV of pH 4.5 for hsTMEM175 WT and mutants. Data are presented as means ± SEM for n ≥ 3. (I) Residues on TM8, TM9, and TM12 that were examined for the regulation of proton conductance. (J) Representative I-V curves of hsTMEM175 mutant H327A, showing the deficiency in K⁺ and proton conductance. (K) Representative I-V curves of hsTMEM175 mutants Q360L and H449A, showing enhancement of K⁺ and proton conductance.