Lipid-Protein Membrane Structure-Function Characterization using Droplet Interface Bilayers

Droplet interface bilayers (DIBs) offer a tunable platform for probing the electromechanical properties of lipid and lipid-peptide membranes under controlled electrical stimulation. DIBs enable both single-channel and ensemble ion conductance measurements over membrane areas orders of magnitude larger than those accessible by traditional patch clamp techniques, thereby allowing membrane-level analyses of electromechanical deformation and its influence on ion-conducting peptides. By systematically tuning membrane structure through the bulk hydrocarbon oil phase (e.g., hexadecane [C16] vs. dodecane/hexadecane [C12/C16] [25%/75%, v/v]), this bottom-up platform enables systematic variation of membrane composition and oil environment, which influence membrane viscoelasticity and structural reorganization, and thereby peptide ion conduction. Detailed procedures are provided for the assembly of gramicidin A-doped 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) membranes using different hydrocarbon oil compositions and for the application of voltage-pulse protocols that drive membranes into metastable electromechanical states. Adaptive membrane ion conduction is characterized, including short-term plasticity-like (STP-like) and long-term potentiation- and depression-like (LTP-like/LTD-like) responses in a model membrane system. More broadly, this protocol provides a robust, reproducible approach for systematically investigating composition-dependent, membrane-level electromechanical contributions to synaptic-like conductive behavior and for understanding how lipid membrane environments modulate ion channel function.