Human Serotonin Receptor 5-HT_1A Preferentially Segregates to the Liquid Disordered Phase in Synthetic Lipid Bilayers

Abstract

We demonstrate successful incorporation of the G protein coupled receptor 5-HT1A into giant unilamellar vesicles using an agarose rehydration method. With direct observation using fluorescence techniques, we report preferential segregation of 5-HT1A into the cholesterol-poor liquid disordered phase of the membrane, contradicting previous reports of lipid raft segregation. Furthermore, altering the concentration of cholesterol and sphingomyelin in ternary mixtures does not alter 5-HT1A segregation into the liquid disordered phase.

Hydrogel-assisted functional reconstitution of human P-glycoprotein (ABCB1) in giant liposomes

“…Since the film of agarose provided partial hydration, we hence hypothesized that such a film of agarose might be useful for reconstituting membrane proteins into giant proteoliposomes. The Malmstadt group has recently reported an elegant approach to form giant proteoliposomes that used direct dilution of detergent-solubilized membrane proteins in combination with lipid hydration from an agarose film [25,26]. Here, we explored whether hydrogel films might also be useful for the formation of giant proteoliposomes from small proteoliposomes since small proteoliposomes are one of the most commonly used membrane protein preparations.…”

Self‐Assembly of Giant Unilamellar Vesicles by Film Hydration Methodologies

“…By modulating the phospholipid to cholesterol ratio, the aquaporin selectively segregated in distinct domains, mimicking cell membrane organization ( Figure 4 ). Using the same protocol, a human serotonin G protein coupled receptor (GPCR) was controllably segregated in liquid disordered phase in liposomes GUVs . GPCRs are omnipresent in human cells, mediating extracellular signals for physiological signaling.…”

G Protein-Coupled Receptors Incorporated into Rehydrated Diblock Copolymer Vesicles Retain Functionality

“…We utilize the agarose hydration method that we have previously reported for the incorporation of 5‐HT 1A R into giant unilamellar vesicles (Figures S1 and S2, Supporting Information) . We incorporate membrane preparations of 5‐HT 1A R with associated G proteins into polymeric membranes made of polybutadiene‐ b ‐poly(ethylene oxide) (PBd(650)‐PEO(400)) at a polymer‐to‐protein molar ratio of 100:1.…”

“…We further exploit the stability of polymersomes and show that following lyophilization and rehydration, diblock copolymer giant unilamellar protein-vesicles (pGUPs) with integrated GPCRs retain vesicle integrity and protein function.We utilize the agarose hydration method that we have previously reported for the incorporation of 5-HT 1A R into giant unilamellar vesicles (Figures S1 and S2, Supporting Information). [14][15][16] We incorporate membrane preparations of 5-HT 1A R with associated G proteins into polymeric membranes made of polybutadiene-b -poly(ethylene oxide)…”