Sourav Ganguly

Chronic Cholesterol Depletion Using Statin Impairs the Function and Dynamics of Human Serotonin1A Receptors

Statins are potent inhibitors of HMG-CoA reductase, the key rate-limiting enzyme in cholesterol biosynthesis, and are some of the best selling drugs globally. We have explored the effect of chronic cholesterol depletion induced by mevastatin on the function of human serotonin(1A) receptors expressed in CHO cells. An advantage with statins is that cholesterol depletion is chronic which mimics physiological conditions. Our results show a significant reduction in the level of specific ligand binding and G-protein coupling to serotonin(1A) receptors upon chronic cholesterol depletion, although the membrane receptor level is not reduced at all. Interestingly, replenishment of mevastatin-treated cells with cholesterol resulted in the recovery of specific ligand binding and G-protein coupling. Treatment of cells expressing serotonin(1A) receptors with mevastatin led to a decrease in the diffusion coefficient and an increase in the mobile fraction of the receptor, as determined by fluorescence recovery after photobleaching measurements. To the best of our knowledge, these results constitute the first report describing the effect of chronic cholesterol depletion on the organization and function of a G-protein-coupled neuronal receptor. Our results assume significance in view of recent reports highlighting the symptoms of anxiety and depression in humans upon statin administration, and the role of serotonin(1A) receptors in anxiety and depression.

Organization of Higher-Order Oligomers of the Serotonin1A Receptor Explored Utilizing Homo-FRET in Live Cells

The serotonin₁(A) receptor is a representative member of the GPCR superfamily and serves as an important drug target. The possible role of GPCR oligomerization in receptor function is an active area of research. We monitored the oligomerization state of serotonin₁(A) receptors using homo-FRET and fluorescence lifetime measurements. Homo-FRET is estimated by a reduction in fluorescence anisotropy and provides a superior approach for exploring oligomerization. In addition, homo-FRET offers the possibility of detecting higher-order oligomers. On the basis of an observed increase in fluorescence anisotropy upon progressive photobleaching and analysis of the difference between the extrapolated anisotropy and the predicted anisotropy of an immobile monomer, we propose the presence of constitutive oligomers of the serotonin₁(A) receptor. To the best of our knowledge, these results constitute the first report of higher-order oligomers for the serotonin₁(A) receptor. We further show that cholesterol depletion and antagonist treatment result in a reduced population of higher-order oligomers. In contrast, agonist stimulation and destabilization of the actin cytoskeleton lead to an increased contribution from higher oligomers. These results provide novel insight into the oligomerization status of the serotonin₁(A) receptor that could enhance the ability to design better therapeutic strategies to combat diseases related to malfunctioning of GPCRs.

Cholesterol Depletion Mimics the Effect of Cytoskeletal Destabilization on Membrane Dynamics of the Serotonin1A Receptor: A zFCS Study

Single-point fluorescence correlation spectroscopy (FCS) of membrane-bound molecules suffers from a number of limitations leading to inaccurate estimation of diffusion parameters. To overcome such problems and with the overall goal of addressing membrane heterogeneities, we performed z-scan FCS (zFCS) of the serotonin(1A) receptor. We analyzed the results according to FCS diffusion laws that provide information on the organization of the diffusing species. Analysis of our results shows that the diffusion coefficients of the receptor and a fluorescently labeled phospholipid are similar when probed at length scales approximately 210 nm. We discuss the significance of the spatiotemporal evolution of dynamics of membrane-bound molecules in the overall context of membrane domains and heterogeneity. Importantly, our results show that the serotonin(1A) receptor exhibits confinement in cell membranes, possibly due to interaction with the actin cytoskeleton. Surprisingly, depletion of membrane cholesterol appears to reduce receptor confinement in a manner similar to that observed in the case of cytoskeletal destabilization, implying possible changes in the actin cytoskeleton induced upon cholesterol depletion. These results constitute the first report on G-protein-coupled receptor dynamics utilizing a combination of zFCS and the FCS diffusion laws, and present a convenient approach to explore cell membrane heterogeneity at the submicron level.

Actin Cytoskeleton-Dependent Dynamics of the Human Serotonin1A Receptor Correlates with Receptor Signaling

Analyzing the dynamics of membrane proteins in the context of cellular signaling represents a challenging problem in contemporary cell biology. Lateral diffusion of lipids and proteins in the cell membrane is known to be influenced by the cytoskeleton. In this work, we explored the role of the actin cytoskeleton on the mobility of the serotonin(1A) (5-HT(1A)) receptor, stably expressed in CHO cells, and its implications in signaling. FRAP analysis of 5-HT(1A)R-EYFP shows that destabilization of the actin cytoskeleton induced by either CD or elevation of cAMP levels mediated by forskolin results in an increase in the mobile fraction of the receptor. The increase in the mobile fraction is accompanied by a corresponding increase in the signaling efficiency of the receptor. Interestingly, with increasing concentrations of CD used, the increase in the mobile fraction exhibited a correlation of approximately 0.95 with the efficiency in ligand-mediated signaling of the receptor. Radioligand binding and G-protein coupling of the receptor were found to be unaffected upon treatment with CD. Our results suggest that signaling by the serotonin(1A) receptor is correlated with receptor mobility, implying thereby that the actin cytoskeleton could play a regulatory role in receptor signaling. These results may have potential significance in the context of signaling by GPCRs in general and in the understanding of GPCR-cytoskeleton interactions with respect to receptor signaling in particular.

Fixation alters fluorescence lifetime and anisotropy of cells expressing EYFP-tagged serotonin1A receptor

Fluorescence microscopic approaches represent powerful techniques to monitor molecular interactions in the cellular milieu. Measurements of fluorescence lifetime and anisotropy enjoy considerable popularity in this context. These measurements are often performed on live as well as fixed cells. We report here that formaldehyde-induced cell fixation introduces heterogeneities in the fluorescence emission of serotonin(1A) receptors tagged to enhanced yellow fluorescent protein, and alters fluorescence lifetime and anisotropy significantly. To the best of our knowledge, our results constitute the first report on the effect of formaldehyde fixation on fluorescence parameters of cellular proteins. We conclude that fluorescence parameters derived from fixed cells should be interpreted with caution.

Differential dynamics of membrane proteins in yeast.

Lateral diffusion of lipids and proteins in yeast plasma membranes has been reported to be anomalously slow, and implicated as a possible reason for polarization in yeast. In order to gain insight into the observed slow diffusion in yeast membranes, we explored lateral diffusion of two proteins of different origin. We compared lateral dynamics of the Candida drug resistance protein-1 (Cdr1p), and the human serotonin(1A) receptor (5-HT(1A)R) by fluorescence recovery after photobleaching (FRAP). Our results show that while Cdr1p-GFP displays slow diffusion, the diffusion of 5-HT(1A)R-EYFP is significantly faster. Interestingly, upon ergosterol depletion, the mobility of Cdr1p-GFP did not exhibit appreciable change, while 5-HT(1A)R-EYFP mobility showed an increase. On the other hand, upon actin cytoskeleton destabilization, the mobile fraction of 5-HT(1A)R-EYFP showed considerable increase, while the mobility of Cdr1p-GFP was not altered. Our results represent the first report on the dynamics of the important drug resistance protein Cdr1p and provide novel insight on diffusion of membrane proteins in yeast membranes.

Metabolic depletion of sphingolipids enhances the mobility of the human serotonin1A receptor.

Sphingolipids are essential components of eukaryotic cell membranes. We recently showed that the function of the serotonin(1A) receptor is impaired upon metabolic depletion of sphingolipids using fumonisin B(1) (FB(1)), a specific inhibitor of ceramide synthase. Serotonin(1A) receptors belong to the family of G-protein coupled receptors and are implicated in the generation and modulation of various cognitive, behavioral and developmental functions. Since function and dynamics of membrane receptors are often coupled, we monitored the lateral dynamics of the serotonin(1A) receptor utilizing fluorescence recovery after photobleaching (FRAP) under these conditions. Our results show an increase in mobile fraction of the receptor upon sphingolipid depletion, while the diffusion coefficient of the receptor did not exhibit any significant change. These novel results constitute the first report on the effect of sphingolipid depletion on the mobility of the serotonin(1A) receptor. Our results assume greater relevance in the broader context of the emerging role of receptor mobility in understanding cellular signaling.

Comparative Analysis of Calcium Spikes upon Activation of Serotonin1A and Purinergic Receptors

Calcium signaling represents one of the most important signaling cascades in cells and regulates diverse processes such as exocytosis, muscle contraction and relaxation, gene expression and cell growth. G protein-coupled receptors (GPCRs) are the most important family of receptors that activate calcium signaling. Since calcium signaling regulates a large number of physiological responses, it is intriguing that how changes in cytosolic calcium levels by a wide range of stimuli lead to signal-specific physiological responses in the cellular interior. In order to address this issue, we have analyzed temporal calcium profiles induced by two GPCRs, the serotonin1A and purinergic receptors. In this work, we have described a set of parameters for the analysis of calcium transients that could provide novel insight into mechanisms responsible for maintaining signal specificity by shaping calcium transients. An interesting feature of calcium signaling that has emerged from our analysis is that the profile of individual transients in a calcium response could play an important role in maintaining downstream signal specificity. In summary, our analysis offers a novel approach to identify differences in calcium response patterns induced by various stimuli.