Spontaneous Depletion of Valinomycin-Mediated Ion Current in Droplet Interface Bilayers

Valinomycin (Val) is a selective ion carrier widely used to develop potassium-selective membranes and study carrier-mediated transport across lipid bilayers. Using either black lipid membranes (BLMs) or supported lipid bilayers (SLBs), previous studies explored valinomycin's selectivity and transport mechanism and quantified the amount of potassium ion current per carrier molecule. Herein, we demonstrate that Val-doped membranes exhibit distinct kinetic behavior when incorporated into droplet interface bilayers (DIBs), a platform known for forming highly resistive membranes ideal for studying ion transport and gating phenomena. Specifically, when Val is reconstituted into DIBs to form potassium-selective membranes, we observe a significant decrease in the voltage-driven ion current over time. We investigate this behavior and quantify the effects of applied voltage, Val placement, and oil type on the resulting ionic current response, explain the underlying causes, and propose a strategy to mitigate the current decay challenge. Using a combination of electrophysiology and UV absorbance measurements, our findings reveal that the valinomycin-mediated ion current is diminished due to the loss of Val from the interior of both the lipid membrane and aqueous droplet via its passive diffusion and partitioning into the surrounding oil phase. Increasing the ionophore concentration, applying a DC voltage, and choosing an oil that is readily retained in the bilayer membrane all quicken the onset and increase the rate of Val depletion. Nonetheless, allowing lipid-coated droplets to incubate for extended periods of time in an oil containing Val leads to a Val-doped DIB with a significantly more stable ionic conductance. More broadly, these behaviors demonstrate a challenge to and a possible solution for using DIBs to study hydrophobic membrane-active species.