Fig. 3

Lithium effect on SNARE-mediated vesicular fusion on suspended membrane. (A) SNARE proteins are the vSNARE, VAMP2, with a single helical domain, located on the vesicle (green) and the tSNARE, a preassembled Sytaxin1A/SNAP25 dimer with a three helices bundle, located on the target membrane (red, yellow and grey). Upon encountering, vSNARE and tSNARE hybridize in a four-helix coil-coiled called a SNAREpin (second panel). The SNAREpin provides energy upon zippering, facilitating fusion pore nucleation (third panel). In the case of fusion mediated by a low SNAREpin number, the fusion pore is transient and reseals after opening (fourth panel). (B) Scheme of the suspended membrane experimental conditions. On the left, the leaflet compositions for the intracellular equivalent, composed of DOPC:DOPS:DOPE:Cholesterol:P(4,5)IP₂ (10:12:35:40:3 mol:mol) and the extracellular equivalent, composed of DOPC:DOPS:DOPE:Cholesterol:Sphingomyelin (SM) (20:5:15:40:20 mol:mol). For all experiments, the intracellular equivalent side contain 150 mM KCl while the extracellular equivalent side is composed of a solution with 150 mM of monovalent chloride salt (MCl) with varying amounts of lithium and potassium. On the right, a scheme of the of the experiment with two compartments: an intracellular one (top) and an extracellular one (bottom), separated by the asymmetric suspended membrane. The intracellular compartment contains vesicles functionalized with vSNAREs to perform fusion and α-hemolysin necessary for current measurements. The suspended membrane is functionalized with tSNAREs facing the intracellular compartment. Hence, SNAREpin assembly and fusion occurs in intracellular compartment which contains potassium buffer. The extracellular equivalent side contains buffer of variable lithium concentrations. Thus, lithium interacts with the extracellular leaflet but not with SNAREs. (C) Scheme of the energetic reaction profile for fusion without any SNAREpin is plotted in grey and displays a fusion energy barrier ∆E_a⁰. Upon the action of a SNAREpin, the fusion barrier is lowered by ∆∆E_a^1SNAREpin (orange). In contrast, in the presence of lithium, the fusion energy barrier is increased by ∆∆E_a^Lithium (blue). (D) Experimental measurements of transient fusion frequency (in min^-1) depending on lithium concentration (in mM) in the extracellular equivalent side, plot in logarithmic scale. As lithium concentration increases, fusion frequency decreases with a sharp transition in the millimolar range. (E) Table with the values of fusion frequency in min^-1 with SD (middle column) for each lithium concentration tested. The column on the right presents the fusion frequency efficiency in percent taking as reference the fusion frequency measured without lithium. (F) In presence of lithium in the extracellular equivalent side, the transition from a docked state to a fused state is harder. Lithium is represented by the red sphere binding to the lipid bilayer.