Reconstitution of Proteoliposomes for Phospholipid Scrambling and Nonselective Channel Assays

Abstract

Phospholipid scramblases catalyze the rapid trans-bilayer movement of lipids down their concentration gradients. This process is essential for numerous cellular signaling functions including cell fusion, blood coagulation, and apoptosis. The importance of scramblases is highlighted by the number of human diseases caused by mutations in these proteins. Because of their indispensable function, it is essential to understand and characterize the molecular function of phospholipid scramblases. Powerful tools to measure lipid transport in cells are available. However, these approaches provide limited mechanistic insights into the molecular bases of scrambling. Here we describe in detail an in vitro phospholipid scramblase assay and the accompanying analysis which allows for determination of the macroscopic rate constants associated with phospholipid scrambling. Notably, members of the TMEM16 family of scramblases also function as nonselective ion channels. To better understand the physiological relevance of this channel function as well as its relationship to the scrambling activity of the TMEM16s we also describe in detail an in vitro flux assay to measure nonselective channel activity. Together, these two assays can be used to investigate the dual activities of the TMEM16 scramblases/nonselective channels.

Keywords: Lipid transport; Nonselective channel; Phospholipids; Scramblases; Scrambling; TMEM16.

Fig. 1

Schematic of phospholipid scrambling and ion flux assays. (a) Schematic of the in vitro scramblase assay. Liposomes are reconstituted with NBD-labeled phospholipids (orange) that distribute equally in the two leaflets. Addition of extraliposomal sodium dithionite reduces the NBD fluorophore (black), causing 50% fluorescence loss in protein-free vesicles (top panel). When a scramblase is present (bottom panel), all NBD-phospholipids become exposed to dithionite, resulting in complete loss of fluorescence [7]. (b) Schematic of in vitro flux assay. Liposomes with high internal KCl concentration (300 mM) are buffer exchanged into low (1 mM) external KCl. In the absence of a scramblase (top panels), the KCl gradient is maintained after buffer exchange. The addition of dithionite (*D) disrupts the liposomes and releases the remaining KCl which can be measured with a AgCl electrode. In the presence of an active nonselective channel (bottom panels), the KCl gradient is lost following buffer exchange due to ion flux. The addition of dithionite (*D) disrupts the liposomes and the remaining KCl is released which is measured with a AgCl electrode

Fig. 2

Methods to analyze scrambling assay traces. (a) Normalized fluorescence decay from protein-free liposomes (green) and scramblase proteoliposomes (red, +Ca²⁺ and black, 0 Ca²⁺). * indicates the addition of dithionite to the extracellular solution. (b) Fixed point method of analyzing scrambling assay traces. The dashed line indicates t = 3τ and arrows point to the fluorescence values (F_PF, F_0Ca2+ and F_max) used for the analysis (Eq. 1). For the traces in this example, $F_{\text{PF}}=0.53F_{0{\text{Ca}}^{2+}}=0.4$ and F_max = 0.14 so ΔF = 33%, indicating that removal of Ca²⁺ causes a 66% decrease in scrambling activity. (c) Normalized fluorescence decays fit (blue) to Eqs. 2 and 3 for scramblase proteoliposomes and protein-free vesicles, respectively. The fit of the fluorescence decay of protein-free liposomes determines L_i^PF and γ while that of the proteoliposomes determines f₀, α, and β. Arrows below the red trace denote f₀. Note the gain in dynamical range with the rate constant analysis. For the traces in this example, f₀(+Ca⁺²) = 0.2, α(+Ca²⁺)~β(+Ca²⁺)~0.1 s⁻¹, while α(0 Ca²⁺)~β(0 Ca²⁺)~0.001 s⁻¹ and f₀(0 Ca²⁺) was constrained to be equal to the value in +Ca²⁺, after verifying that the reconstitution efficiency is the same in the two conditions. Note that by fitting the complete trace to Eq. 2 the decrease in activity is ~100-fold, which reflects the dramatic slowdown of the scrambling process visible in the raw data

Fig. 3

Flux assay analysis. Raw trace for flux assay of protein-free liposomes (black) and proteoliposomes (red). Points needed for analysis are marked with arrows: V₀ is the beginning of the trace and measures the buffer before calibration; V_cal is measured after the addition of 18 μL of 10 mM KCl to calibrate the system (indicated by #); V_lipo is measured after the addition of liposomes (indicated by ^) and V_tot is the signal after the addition of detergent (indicated by *). This is the measure of the trapped Cl⁻ from inside the liposomes. For the traces in this example, ΔCl(Protein-free) 0.49 mM, and ΔCl(Proteoliposomes) = 0.16 mM, so that A = 0.67, indicating that ~67% of the channel liposomes contain at least one active nonselective channel