Investigating the interactions of the 18kDa translocator protein and its ligand PK11195 in planar lipid bilayers

Abstract

The functional effects of a drug ligand may be due not only to an interaction with its membrane protein target, but also with the surrounding lipid membrane. We have investigated the interaction of a drug ligand, PK11195, with its primary protein target, the integral membrane 18kDa translocator protein (TSPO), and model membranes using Langmuir monolayers, quartz crystal microbalance with dissipation monitoring (QCM-D) and neutron reflectometry (NR). We found that PK11195 is incorporated into lipid monolayers and lipid bilayers, causing a decrease in lipid area/molecule and an increase in lipid bilayer rigidity. NR revealed that PK11195 is incorporated into the lipid chain region at a volume fraction of ~10%. We reconstituted isolated mouse TSPO into a lipid bilayer and studied its interaction with PK11195 using QCM-D, which revealed a larger than expected frequency response and indicated a possible conformational change of the protein. NR measurements revealed a TSPO surface coverage of 23% when immobilised to a modified surface via its polyhistidine tag, and a thickness of 51Å for the TSPO layer. These techniques allowed us to probe both the interaction of TSPO with PK11195, and PK11195 with model membranes. It is possible that previously reported TSPO-independent effects of PK11195 are due to incorporation into the lipid bilayer and alteration of its physical properties. There are also implications for the variable binding profiles observed for TSPO ligands, as drug-membrane interactions may contribute to the apparent affinity of TSPO ligands.

Keywords: 1,2-dimyristoyl-sn-glycero-3-phosphocholine; 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine; 1-(2-Chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide; ANTA; Bmax; CM4; CMAu; CMSi; DDM; DMPC; DMPE; DTSP; DTT; E. coli; Escherichia coli; IPTG; Kd; Lipid bilayer; Membrane protein; NR; NSAIDs; NTA; Neutron reflectometry; OD; PBS; PC; PK11195; QCM-D; SDS; SLD; SPR; SUV; TEV; TSPO; TspO; affinity or equilibrium dissociation constant; dithiobis(succinimidyl propionate); dithiothreitol; gold contrast matched water; isopropyl β-d-1-thiogalactopyranoside; maximal number of receptor binding sites; n-dodecyl-β-d-maltopyranoside; neutron reflectometry; nitrilotriacetic acid; nitrilotriacetic acid group with a terminal amino group; non-steroidal anti-inflammatory drugs; optical density; phosphate buffered saline; phosphatidylcholine; quartz crystal microbalance with dissipation monitoring; scattering length density; scattering length density 4 contrast matched water; silicon contrast matched water; small unilamellar vesicle; sodium dodecyl sulphate; surface plasmon resonance; tobacco etch virus; translocator protein; tryptophan rich sensory protein.