Free cholesterol alters lipid raft structure and function regulating neutrophil Ca2+ entry and respiratory burst:...

Recent studies associate cholesterol excess and atherosclerosis with inflammation. The link between these processes is not understood, but cholesterol is an important component of lipid rafts. Rafts are thought to concentrate membrane signaling molecules and thus regulate cell signaling through G protein-coupled pathways. We used methyl beta-cyclodextrin to deplete cholesterol from polymorphonuclear neutrophil (PMN) rafts and thus study the effects of raft disruption on G protein-coupled Ca(2+) mobilization. Methyl beta-cyclodextrin had no effect on Ca(2+) store depletion by the G protein-coupled agonists platelet-activating factor or fMLP, but abolished agonist-stimulated Ca(2+) entry. Free cholesterol at very low concentrations regulated Ca(2+) entry into PMN via nonspecific Ca(2+) channels in a biphasic fashion. The specificity of cholesterol regulation for Ca(2+) entry was confirmed using thapsigargin studies. Responses to cholesterol appear physiologic because they regulate respiratory burst in a proportional biphasic fashion. Investigating further, we found that free cholesterol accumulated in PMN lipid raft fractions, promoting formation and polarization of membrane rafts. Finally, the transient receptor potential calcium channel protein TRPC1 redistributed to raft fractions in response to cholesterol. The uniformly biphasic relationships between cholesterol availability, Ca(2+) signaling and respiratory burst suggest that Ca(2+) influx and PMN activation are regulated by the quantitative relationships between cholesterol and other environmental lipid raft components. The association between symptomatic cholesterol excess and inflammation may therefore in part reflect free cholesterol- dependent changes in lipid raft structure that regulate immune cell Ca(2+) entry. Ca(2+) entry-dependent responses in other cell types may also reflect cholesterol bioavailability and lipid incorporation into rafts.