Interplay of Cholesterol and Actin in Neurotransmitter GPCR Signaling: Insights from Chronic Cholesterol Depletion Using Statin

Serotonin1A receptors are important neurotransmitter receptors in the G protein-coupled receptor (GPCR) family and modulate a variety of neurological, behavioral, and cognitive functions. We recently showed that chronic cholesterol depletion by statins, potent inhibitors of HMG-CoA reductase (the rate-limiting enzyme in cholesterol biosynthesis), leads to polymerization of the actin cytoskeleton that alters lateral diffusion of serotonin1A receptors. However, cellular signaling by the serotonin1A receptor under chronic cholesterol depletion remains unexplored. In this work, we explored signaling by the serotonin1A receptor under statin-treated condition. We show that cAMP signaling by the receptor is reduced upon lovastatin treatment due to reduction in cholesterol as well as polymerization of the actin cytoskeleton. To the best of our knowledge, these results constitute the first report describing the effect of chronic cholesterol depletion on the signaling of a G protein-coupled neuronal receptor. An important message arising from these results is that it is prudent to include the contribution of actin polymerization while analyzing changes in membrane protein function due to chronic cholesterol depletion by statins. Notably, our results show that whereas actin polymerization acts as a negative regulator of cAMP signaling, cholesterol could act as a positive modulator. These results assume significance in view of reports highlighting symptoms of anxiety and depression in humans upon statin administration and the role of serotonin1A receptors in anxiety and depression. Overall, these results reveal a novel role of actin polymerization induced by chronic cholesterol depletion in modulating GPCR signaling, which could act as a potential therapeutic target

Identification of Cholesterol Binding Sites in the Serotonin_1A Receptor

The serotonin_1A receptor is a representative member of the G protein-coupled receptor (GPCR) superfamily and serves as an important drug target in the development of therapeutic agents for neuropsychiatric disorders. Previous work has shown the requirement of membrane cholesterol in the organization, dynamics, and function of the serotonin_1A receptor. We show here that membrane cholesterol binds preferentially to certain sites on the serotonin_1A receptor by performing multiple, long time scale MARTINI coarse-grain molecular dynamics simulations. Interestingly, our results identify the highly conserved cholesterol recognition/interaction amino acid consensus (CRAC) motif on transmembrane helix V as one of the sites with high cholesterol occupancy, thereby confirming its role as a putative cholesterol binding motif. These results represent the first direct evidence for membrane cholesterol binding to specific sites on the serotonin_1A receptor and represent an important step in our overall understanding of GPCR function in health and disease

Calcium response in CHO-5-HT_1AR cells upon stimulation with serotonin or ATP.

<p>Panel (A) shows successive images of calcium response in cells upon stimulation with serotonin acquired with a time interval of 7.9 sec. CHO-5-HT_1AR cells were loaded with Fluo-3/AM to monitor changes in cytosolic calcium concentration. Time-lapse imaging was performed with a 20×/0.75 NA objective. The scale bar (shown in top left panel) represents 50 µm. Similar response was observed when cells were stimulated with ATP. Representative temporal profiles of calcium response induced by either (B) serotonin or (C) ATP are shown. The concentration of serotonin used was 100 µM and that of ATP was 500 nM, respectively. Both serotonin and ATP induce the occurrence of multiple spikes consisting of rise and decay phases. A typical calcium spike with rise and decay phases is shown in the inset in panel (B). The temporal profile of calcium response was intensity normalized such that the maximum and minimum (basal) intensities corresponded to values 2 and 1, respectively. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051857#s2" target="_blank">Materials and Methods</a> for more details.</p

Calcium responses induced by ATP.

<p>Representative (A) distinct or (B) overlapping calcium spikes observed upon stimulation with 500 nM ATP. The propensity of the occurrence of overlapping calcium spikes increased with increase in ATP concentration. The temporal profile of calcium response was intensity-normalized such that the maximum and minimum (basal) intensities corresponded to values 2 and 1, respectively. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051857#s2" target="_blank">Materials and Methods</a> for more details.</p

The rate of increase in cytosolic calcium levels depends on ligand concentration.

<p>The figure shows the apparent time constant for the rise phase of the first calcium spike induced with varying concentrations of either (A) serotonin or (B) ATP. R² values for fitting of the rise phase of calcium spikes with monoexponential function range from 0.94–1 in all cases. Data represent means ± SEM of more than 30 cells from at least four independent experiments. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051857#s2" target="_blank">Materials and Methods</a> for more details.</p

The maximum fold change in cytosolic calcium levels depends on ligand concentration.

<p>The figure shows calcium response in terms of maximum fold change in cytosolic calcium levels induced with either (A) serotonin or (B) ATP. Only the first spike of calcium response (when composed of multiple spikes) induced by ligands was analyzed. Data represent means ± SEM of more than 30 cells from at least four independent experiments. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051857#s2" target="_blank">Materials and Methods</a> for more details.</p

Dose response plots for calcium signaling induced by ligands in CHO-5-HT_1AR cells.

<p>The figure shows calcium response in terms of a number of calcium spikes visualized per cell induced with either (A) serotonin or (B) ATP. The curves are nonlinear regression fits to the experimental data using <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051857#pone.0051857.e001" target="_blank">eqn. 1</a>. Data represent means ± SEM of more than 30 cells from at least four independent experiments. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051857#s2" target="_blank">Materials and Methods</a> for more details.</p

Amplitude, full width at half maxima (FWHM) and area vary for individual spike in a multi-spike response.

<p>The amplitude of calcium spike represents the maximum fold change in cytosolic calcium level. The area of calcium spike corresponds to the amount of calcium released in the cytoplasm and FWHM denotes the life span of the calcium spike in the cytoplasm. Panels (A), (B) and (C) show amplitude, FWHM and area for serotonin-induced calcium spikes (blue triangle), and ATP-induced distinct (red triangle) and overlapping (•) calcium spikes, respectively. The temporal profile of calcium response was intensity-normalized such that the maximum and minimum (basal) intensities corresponded to values 2 and 1, respectively, before fitting it with a multi-gaussian function. In case of serotonin and ATP-stimulated calcium spikes, R² values for fitting of calcium spikes with Gaussian function ranged from 0.92–0.98 and 0.94–0.99, respectively. Values represent means ± SEM of more than 50 cells from at least four independent experiments. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051857#s2" target="_blank">Materials and Methods</a> for more details.</p

Depth-Dependent Heterogeneity in Membranes by Fluorescence Lifetime Distribution Analysis

Biological membranes display considerable anisotropy due to differences in composition, physical characteristics, and packing of membrane components. In this Letter, we have demonstrated the environmental heterogeneity along the bilayer normal in a depth-dependent manner using a number of anthroyloxy fatty acid probes. We employed fluorescence lifetime distribution analysis utilizing the maximum entropy method (MEM) to assess heterogeneity. Our results show that the fluorescence lifetime heterogeneity varies considerably depending on fluorophore location along the membrane normal (depth), and it is the result of the anisotropic environmental heterogeneity along the bilayer normal. Environmental heterogeneity is reduced as the reporter group is moved from the membrane interface to a deeper hydrocarbon region. To the best of our knowledge, our results constitute the first experimental demonstration of anisotropic heterogeneity in bilayers. We conclude that such graded environmental heterogeneity represents an intrinsic characteristics of the membrane bilayer and envisage that it has a role in the conformation and orientation of membrane proteins and their function

Organization and Dynamics of Hippocampal Membranes in a Depth-Dependent Manner: An Electron Spin Resonance Study

Organization and dynamics of neuronal membranes represent crucial determinants for the function of neuronal receptors and signal transduction. Previous work from our laboratory has established hippocampal membranes as a convenient natural source for studying neuronal receptors. In this work, we have monitored the organization and dynamics of hippocampal membranes and their modulation by cholesterol and protein content utilizing location (depth)-specific spin-labeled phospholipids by ESR spectroscopy. The choice of ESR spectroscopy is appropriate due to slow diffusion encountered in crowded environments of neuronal membranes. Analysis of ESR spectra shows that cholesterol increases hippocampal membrane order while membrane proteins increase lipid dynamics resulting in disordered membranes. These results are relevant in understanding the complex organization and dynamics of hippocampal membranes. Our results are significant in the overall context of membrane organization under low cholesterol conditions and could have implications in neuronal diseases characterized by low cholesterol conditions due to defective cholesterol metabolism