Lipid Profile and Functional Characterization of the Human α4 β2 Nicotinic Acetylcholine Receptor Detergent Complexes

The human a4 b2 nicotinic acetylcholine receptor (nAChR) is the most expressed nAChR in the human brain, and it is known to be directly linked to nicotine addiction, and common neurodegenerative pathologies in the US like schizophrenia, and Alzheimer’s disease. It is known that the localization and function of membrane proteins like the a4 b2 nAChR is related to membrane thickness, and the lipid environment of the protein-lipid interface. In consequence, detergents used in membrane protein solubilization often abolish the receptor’s function by altering the surrounding lipid composition. Recent studies characterized the function of detergent-solubilized nAChR from the electric tissue of Torpedo californica (Tc) using the two-electrode voltage clamp (TEVC) technique. Results showed that most common detergents used in membrane protein extraction, abolish the receptor’s function, however, phospholipid analog detergents (PADs) like lysofoscholine (LFC) 14, and 16, produced nAChR-detergent complexes (nAChR-DCs) that preserved the receptor’s function after solubilization. This led to the analysis of the lipid environment of the nAChR-DCs, which suggested a strong correlation between detergent delipidation and the function of solubilized nAChR. Although, the first high definition structure of the a4 b2 nAChR was recently published, the nAChR-DCs used for crystallization were not assayed for function after detergent solubilization. Currently, there is no available evidence to confirm that a4 b2 nAChR-DCs extracted from transfected HEK cells are functional upon their use in crystallization trials.

Our long-term goal is to study and understand the protein-lipid interactions that are essential for nAChRs to remain functional after solubilization, and ultimately elucidate their high resolution three-dimensional structures. Compared to common detergents, PAD-solubilized nAChR from Tc suggests a strong link between higher phospholipid/nAChR ratios, and preserved receptor function. The overall objective of this project is to assess the lipid composition of detergent solubilized a4 b2 nAchR-DCs expressed in HEK GnTI- cells to correlate detergent delipidation with loss of receptor function, and produce functional preparations suitable for crystallization. We hypothesize that solubilizing the expressed a4 b2-nAChR using PADs like LFC-16, and LFC-14 will preserve their function due to reduced delipidation of essential lipids. In addition, the resulting a4 b2 nAchR-DCs will be used for crystallization trials using vapor diffusion, and modified lipidic cubic phase (LCP) methods to screen for diffractable protein crystals.