Pharmacological tools to modulate ordered membrane domains and order-dependent protein function

Ordered membrane nanodomains colloquially known as "lipid rafts" have many proposed cellular functions. However, pharmacological tools to modulate protein affinity for rafts and to manipulate raft formation are currently lacking. We screened 24,000 small molecules for compounds that impact the raft affinity of a known raft-preferring model protein, peripheral myelin protein 22 (PMP22), in giant plasma membrane vesicles (GPMVs). Hits were tested against another model raft protein, MAL, and also tested for their impact on raft stability. We identified three chemically distinct tools for manipulating lipid rafts. Two compounds were found to destabilize ordered domains (VU0607402 and VU0519975) while a third (primaquine diphosphate) increased PMP22 partitioning and stabilized ordered domains. While discovered in a PMP22-focused screen, all three were seen to modulate raft formation in a protein-independent manner by altering lipid-lipid interactions and membrane fluidity. Acute treatment of live cells with the raft destabilizing compound, VU0607402 was seen to modulate TRPM8 channel function, highlighting the utility of this compound in live-cell experiments for dissecting the role that membrane order and fluidity play in cell signaling. These compounds provide pharmacological tools for probing lipid raft properties and function in biophysical experiments and in living cells.