Cell-free expression of synthetic PKD2L1 protein and incorporation into lipid vesicles.

A) Schematic of cell-free protein expression into synthetic lipid vesicles and subsequent electroformation with Vesicle Prep Pro (Nanion). B) Full length PKD2L1-GFP protein detected by Western Blot after cell-free expression into vesicles. C) Monitored fluorescence over time of cell-free expressed PKD2L1-GFP and a non-fluorescent control plasmid produced in the presence or absence of lipid vesicles.

Orientation of synthetic PKD2L1 channels in vesicles.

A) Schematic of possible ion channel orientation outcomes from PKD2L1 cell-free expression (top) and hypothesized fluorescence results when Cell488 and Surface647 added (bottom). B) Fluorescent confocal images from the SNAP-tagged vesicles. The scale bar represents 10 μm for all images. C) Vesicle percentage depicts the percent of vesicles with each fluorescent output, (N=65 vesicles).

Synthetic PKD2L1 channels are functional in artificial membranes.

A) Images of voltage clamped GUVs with incorporated PKD2L1-GFP channels. Left, establishing high-resistance seals in the on-cell patch configuration. Right, transitioning to the inside-our patch configuration. Scale bar = 20 μm. B) Example unitary single channel current records from GUVs reconstituted with or without PKD2L1 protein. Vesicles were patched using the symmetrical 150 mM K+ conditions (see methods) and PKD2L1 single channel current producing full and sub-conductances are colored black and blue, respectively. C) Average single-channel current amplitudes. Conductance (γ) estimated by fitting the average single channel currents to a linear equation. Error (grey) indicates SEM from N=3-8 GUVs. Several replicates lacked single channel openings at all test potentials. Conductance estimates from individual GUVs are shown in Figure 3—Supplement Figure 1A. D) PKD2L1 single channel current recorded using asymmetric cationic solutions, with 150 mM K+ in the bath and 150 mM NMDG+ in the pipette. E) Resulting average single-channel current amplitudes where no inward single channel current was detected (N=3-4 GUVs).

Conductance properties of polycystins measured from GUV and cilia membranes.

Functional synthetic PKD2 channels in artificial membranes.

A) Example unitary single channel current records from GUVs reconstituted with PKD2 protein in symmetrical 150 mM K+ conditions producing full (black traces) and sub-conductances (blue traces), respectively. B, C) Average single-channel current amplitudes recorded using K+ or NMDG+ in the recording electrode solution. Conductance (γ) estimated by fitting the average single channel currents to a linear equation. Error (grey) indicate SEM from K+ (N=3-12 GUVs) and NMDG+ (N=2-5 GUVs) conditions.