Fig. 4: Asymmetric liposome stopped-flow Tl⁺ flux assay.

a Schematic representation of asymmetric liposome stopped-flow Tl⁺ flux assay (created with BioRender.com). Starting with 3:1 POPC:POPE liposomes reconstituted with ELIC, the sample was either treated with mβCD (top, “No Exchange”), or treated with mβCD/POPG to introduce 25 mole% POPG to the outer leaflet (bottom, “Exchange”). Right: Representative fluorescence quenching traces for no exchange (top) and exchange (bottom) samples showing control (no agonist) and responses to 10 mM propylamine with 500 ms and 10 s delay times. b Zeta potential measurements of symmetric 3:1 POPC:POPE liposomes (3:1 PC:PE) (n = 3), symmetric 2:1:1 POPC:POPE:POPG liposomes (2:1:1) (n = 3), asymmetric 3:1 POPC:POPE liposomes treated to introduce 25 mol% POPG to the outer leaflet (3:1 PC:PE Ex) (n = 2), and asymmetric 3:1 POPC:POPE proteoliposomes with ELIC treated to introduce 25 mol% POPG to the outer leaflet (3:1 PC:PE Ex ELIC) (n = 4). c Tl⁺ flux rates of WT ELIC in 3:1 PC:PE no exchange liposomes (red, “No Ex”) and exchanged asymmetric liposomes where 25 mol% POPG is introduced to the outer leaflet (black, “Ex”) (n = 4). Channel activation was elicited by 10 mM propylamine. d Weighted time constant of desensitization in the presence of 10 mM propylamine from Tl⁺ flux assay comparing no exchange and exchange conditions (n = 4). Statistical analysis was performed using a paired, two-sided T-test. e Peak rate of Tl+ flux in response to 10 mM propylamine in no exchange and exchange conditions (n = 4). Statistical analysis was performed using a paired, two-sided T-test. All data are shown as mean ± s.e. for (n) independent experiments. Source data are provided as a source data file.