Stereospecific requirement of cholesterol in the function of the serotonin1A receptor

Abstract

The serotonin1A receptor is an important member of the G protein-coupled receptor (GPCR) family. It is involved in the generation and modulation of a variety of cognitive and behavioral functions, and serves as a drug target. Previous work from our laboratory has established the sensitivity of the function of the serotonin1A receptor to membrane cholesterol. Solubilization of the hippocampal serotonin1A receptor utilizing the zwitterionic detergent CHAPS is accompanied by loss of cholesterol and results in reduction in specific ligand binding. Replenishment of cholesterol to solubilized membranes restores specific ligand binding to the receptor. We utilized this strategy of sterol replenishment of solubilized membranes to explore the stereospecific stringency of cholesterol for receptor function. We used two stereoisomers of cholesterol, ent-cholesterol (enantiomer of cholesterol) and epi-cholesterol (a diastereomer of cholesterol) for this purpose. Importantly, we show here that while ent-cholesterol could replace cholesterol in supporting receptor function, epi-cholesterol could not. These results imply that the requirement of membrane cholesterol for the serotonin1A receptor function is diastereospecific, yet not enantiospecific. Our results extend and help define specificity of the interaction of membrane cholesterol with the serotonin1A receptor, and represent the first report utilizing ent-cholesterol to examine stereospecificity of GPCR-cholesterol interaction.

Molecular rheology of neuronal membranes explored using a molecular rotor: Implications for receptor function

“…An important aspect of our results is that the interaction between cholesterol and the serotonin1A receptor appears to be stringent, since immediate biosynthetic precursors of cholesterol (differing with cholesterol merely in a double bond) were not able to maintain receptor function (Paila et al, 2008;Singh et al, 2009). In addition, we recently showed that the requirement of cholesterol for receptor function is diastereospecific, but not enantiospecific (Jafurulla et al, 2014). This difference in sterol stereospecificity for receptor function appears to be related to membrane dipole potential (Bandari et al, 2014).…”

“…Native hippocampal membranes were prepared from frozen hippocampal tissue, as described previously (Jafurulla et al, 2014). The final pellet (native membranes) was suspended in a minimum volume of buffer C (50 mM Tris, pH 7.4), homogenized using a hand-held Dounce homogenizer, flash frozen in liquid nitrogen and stored at -80 ºC.…”

“…Briefly, membranes resuspended at a protein concentration of ~2 mg/ml were treated with different concentrations of MβCD in buffer C at room temperature (~25 ºC) with constant shaking for 1 h. Membranes were then spun down at 50,000xg for 5 min, washed once with buffer C and resuspended in the same buffer. Cholesterol was estimated using the Amplex Red cholesterol assay kit (Amundson and Zhou, 1999).Radioligand binding assays were carried out as described previously (Jafurulla et al, 2014).Native and cholesterol-depleted hippocampal membranes containing 50 nmol of total phospholipids were suspended in 1.5 ml of buffer C and used for fluorescence anisotropy experiments. The amount of probe added was such that the final probe concentration was 0.25 mol% with respect to the total phospholipid content.…”

GPCRs: Lipid-Dependent Membrane Receptors That Act as Drug Targets

“…Membrane cholesterol has been shown to modulate the function of a number of GPCRs [35,36,[41][42][43][44][66][67][68][69][70][71][72][73][74]. A specific GPCR that has received a lot of attention in terms of cholesterol sensitivity of its organization, dynamics, and function is the serotonin 1A receptor [36,42,43].…”

“…This was demonstrated utilizing a variety of approaches which include (i) physical depletion of membrane cholesterol using the sterol-specific carriers such as methyl--cyclodextrin (M CD) [66,68], (ii) treatment with agents such as nystatin (a polyene antibiotic) [67] and digitonin (a saponin family detergent) [70], which complex cholesterol and modulate the availability of membrane cholesterol without physically depleting it, (iii) enzymatic oxidation of cholesterol to cholestenone by cholesterol oxidase [69], and The results show that the requirement of membrane cholesterol for the serotonin 1A receptor function is diastereospecific but not enantiospecific. Adapted and modified from [35]. See text for more details.…”