Potential Implications of Cholesterol and Phosphatidylinositol 4,5-Bisphosphate (PIP2) Interactions With The Cholesterol-Sensitive AC418W Acetylcholine Receptor Mutation at Lipid Rafts

Abstract

Lipid rafts, specialized membrane microdomains in the plasma membrane that are rich in cholesterol and sphingolipids, are hot-spots for a number of important cellular processes. The novel acetylcholine receptor (AChR) mutation αC418W was shown to be cholesterol-sensitive (Santiago et al., 2001) and to accumulate in microdomains rich in the membrane raft marker protein caveolin-1 (Baez-Pagan et al., 2008). The objective of this study is to gain insight into the mechanism by which lateral segregation into specialized raft membrane microdomains regulates the activatable pool of AChRs. We performed Fluorescent Recovery After Photobleaching (FRAP) experiments and whole-cell patch clamp recordings of GFP-encoding mus musculus AChRs transfected into HEK 293 cells to assess the role of cholesterol levels in the diffusion and functionality of the AChR (WT and αC418W). Our findings support the hypothesis that a cholesterol-sensitive AChR might reside in a specialized membrane microdomain; however, when cholesterol is depleted in vitro or in vivo, the caveolae disrupt and the cholesterol-sensitive AChRs are released to the pool of activatable receptors. Furthermore, our results suggest that phosphatidylinositol 4,5-bisphosphate (PIP2), which is concentrated in lipid rafts, may be responsible for the increase in whole-cell currents observed upon cholesterol depletion for the αC418W AChR mutant.This work was supported by NIH Grants 2RO1GM56371-12 and 2U54NS43011.