Capacitance-based measurement of calcium-mediated vesicle fusion upon microinjection in droplet interface bilayers
Year: 2026
Type: preprint
Abstract: The droplet interface bilayer (DIB) offers a tunable and stable model membrane platform for studying transmembrane proteins (TMPs). Yet, the library of TMPs known to incorporate into DIBs remains limited to those that spontaneously self-insert from aqueous microdroplets. Moreover, incorporating TMP-decorated liposomes into DIB droplets prior to bilayer formation can disrupt lipid assembly and leave TMPs poorly protected from the oil-water interface. We overcome these hurdles by leveraging calcium-mediated vesicle fusion (CMVF) to deliver bilayer-stable TMPs into DIBs via post-bilayer formation microinjection. Departing from prior works, we employed capacitance-based measurements of intrinsic membrane potential to quantify fusion-induced changes in DIB lipid composition. Our results show that CMVF of symmetric, fusogenic liposomes with an asymmetric DIB reduces its transmembrane potential by an amount that corresponds to the fused area of the bilayer: injecting ~40 nL of fusogenic liposomes into one 400 nL droplet resulted in ~15% of the bilayer area becoming less asymmetric in leaflet composition. Moreover, repeated injections produced cumulative vesicle fusion, while omission of fusogenic lipids or Ca2+ ions abolished changes in membrane potential. After quantifying the CMVF process, we used it to enable and enhance the incorporation of multiple TMPs into DIBs. First, injection of proteoliposomes containing TREK-1 proteins, which do not readily insert into DIBs without fusion, consistently produced stochastic, multi-channel gating within minutes. TREK-1 channels exhibited bipolar voltage dependence and pharmacological activation by arachidonic acid, confirming native channel function. Separately, injection of proteoliposomes decorated with OmpF proteins yielded comparable increases in nominal membrane conductance to spontaneously-inserted OmpF proteins, but at half the driving voltage and despite delivering 10X less protein, indicating that fusion incorporates functional TMPs more efficiently than diffusion-limited protein insertion. Together, these results establish capacitance-based measurements and CMVF as a quantitatively verifiable route for improving TMP incorporation into planar lipid bilayers.
Source: ChemRxiv
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W7204765110