Protein reconstitution and 86Rb flux assay

All proteins used in the flux assay were purified in the DMNG detergent and reconstituted into lipid vesicles composed of 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE, 7.5 mg/mL) and 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG, 2.5 mg/mL) at a protein/lipid ratio of 2 μg/mg using the same method as described¹⁵ with the following modifications: 20 mM DM was used to solubilize the lipid and the detergent/lipid/protein mixture were dialyzed against a reconstitution buffer of 10 mM HEPES buffered with 4 mM NMDG, pH 7.4, and 450 mM KCl or NaCl to slowly remove the detergent. The reconstituted liposome samples were kept at -80 °C in 100 μL aliquots.

The ⁸⁶Rb flux assay was performed following the same procedures as described¹⁵. Liposomes were thawed and sonicated in a bath sonicator for 40 seconds before the assay. To remove extra-liposomal salt (NaCl or KCl), 100 μL samples were passed through a pre-spun Sephadex G-50 fine gel filtration column (1.5 ml bed volume in a 5 ml disposable spin column) swollen in 400 mM Sorbitol, 10 mM HEPES buffered with 4 mM NMDG, pH 7.4. 160 μL of liposome samples collected after this buffer exchange step were added to 320 μL ⁸⁶Rb flux buffer (400 mM Sorbitol, 10 mM HEPES buffered with 4 mM NMDG, pH 7.4, 50 μM NaCl or KCl, and 5 μM ⁸⁶RbCl). At desired time points, 60 μL of this reaction mixture were passed through a pre-spun Sephadex G-50 fine gel filtration column as described above to remove extra-liposomal ⁸⁶Rb and stop the flux. The eluate was mixed with 10 mL scintillation cocktail and its radioactivity was measured in a scintillation counter. For time dependent measurement of ⁸⁶Rb influx, the radioactivity of each sample was normalized against the maximum ⁸⁶Rb influx. The maximum ⁸⁶Rb influx for K⁺-loaded liposomes was obtained by adding 1 μg/mL of valinomycin to a 60 μL reaction mixture and allowing the influx to proceed for 2 minutes before sample collection using Sephadex G-50 column. 10 μg/mL of gramicidin A was used to obtain the maximum ⁸⁶Rb influx for Na⁺-loaded liposomes. For competition assay, CmTMEM175-containing liposomes were loaded with KCl and flux was allowed to proceed for 30 minutes before the radioactivity levels in liposomes were measured. The tested ions (Na⁺, K⁺, Rb⁺, Cs⁺ or NMDG⁺) were added directly into the flux buffer, making the final concentrations of 0.1, 0.5 or 1.0 mM after mixing with liposomes, and samples of reaction mixture were collected 30 minutes after the reaction. The radioactivity of each sample in the competition assay was normalized to the reaction mixture without competition ions.