Role of Cholesterol and its Biosynthetic Precursors on Membrane Organization and Dynamics: A Fluorescence Approach.

Cholesterol is the most representative sterol present in membranes of higher eukaryotes, and is the end product of a long and multistep biosynthetic pathway. Lathosterol and zymosterol are biosynthetic precursors of cholesterol in Kandutsch-Russell and Bloch pathways, respectively. Lathosterol differs with cholesterol merely in the position of the double bond in the sterol ring, whereas zymosterol differs with cholesterol in position and number of double bonds. In this work, we have monitored the effect of cholesterol and its biosynthetic precursors (lathosterol and zymosterol) on membrane organization and dynamics in fluid and gel phase membranes. Toward this goal, we have utilized two fluorescent membrane probes, DPH and its cationic derivative TMA-DPH. Our results using these probes show that cholesterol and its biosynthetic precursors (lathosterol and zymosterol) exhibit similar trend in maintaining membrane organization and dynamics (as reported by fluorescence anisotropy and apparent rotational correlation time), in fluid phase POPC membranes. Notably, although lathosterol and zymosterol show similar trend in maintaining membrane organization and dynamics, the corresponding change for cholesterol is different in gel phase DPPC membranes. These results demonstrate that the position and number of double bonds in sterols is an important determinant in maintaining membrane physical properties. Our results assume significance since accumulation of precursors of cholesterol have been reported to be associated with severe pathological conditions.

Role of Cholesterol and Its Immediate Biosynthetic Precursors in Membrane Dynamics and Heterogeneity: Implications for Health and Disease.

Cholesterol is an indispensible component of cellular membranes in higher eukaryotes and plays a vital role in many cellular functions. 7-Dehydrocholesterol (7-DHC) and desmosterol represent two immediate biosynthetic precursors of cholesterol in the Kandutsch-Russell and Bloch pathways of cholesterol biosynthesis, respectively. Although 7-DHC and desmosterol differ from cholesterol merely by a double bond, accumulation of these two immediate biosynthetic precursors due to defective cholesterol biosynthesis leads to severe developmental and neurological disorders. In this context, we explored the role of cholesterol and its immediate biosynthetic precursors (7-DHC and desmosterol) on the dynamics and heterogeneity of fluid phase POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and gel phase DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) membranes, using fluorescence lifetime distribution analysis of Nile Red (9-diethylamino-5H-benzo[α]phenoxazine-5-one) using the maximum entropy method (MEM). We show here that the membrane interfacial dynamic heterogeneity, manifested as the width of the fluorescence lifetime distribution of Nile Red, exhibited by 7-DHC and desmosterol vastly differ from that displayed by cholesterol, particularly in fluid phase membranes. We conclude that a subtle alteration in sterol structure could considerably alter dynamic membrane heterogeneity, which could have implications in pathogenicity associated with defective cholesterol biosynthesis.

Role of glycosphingolipids in the function of human serotonin1A receptors.

Glycosphingolipids are essential components of eukaryotic cell membranes and are involved in the regulation of cell growth, differentiation, and neoplastic transformation. In this work, we have modulated glycosphingolipid levels in CHO cells stably expressing the human serotonin(1A) receptor by inhibiting the activity of glucosylceramide synthase using (±)-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), a commonly used inhibitor of the enzyme. 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. We explored the function of the serotonin(1A) receptor under glycosphingolipid-depleted condition by monitoring ligand-binding activity and G-protein coupling of the receptor. Our results show that ligand binding of the receptor is impaired under these conditions although the efficiency of G-protein coupling remains unaltered. The expression of the receptor at the cell membrane appears to be reduced. Interestingly, our results show that the effect of glycosphingolipids on ligand binding caused by metabolic depletion of these lipids is reversible. These novel results demonstrate that glycosphingolipids are necessary for the function of the serotonin(1A) receptor. We discuss possible mechanisms of specific interaction of glycosphingolipids with the serotonin(1A) receptor that could involve the proposed 'sphingolipid-binding domain'.

Oligomerization of the serotonin(1A) receptor in live cells: a time-resolved fluorescence anisotropy approach.

The serotonin(1A) receptor is a representative member of the G-protein coupled receptor (GPCR) superfamily and serves as an important target in the development of therapeutic agents for neuropsychiatric disorders. Oligomerization of GPCRs is an important contemporary issue since it is believed to be a crucial determinant for cellular signaling. In this work, we monitored the oligomerization status of the serotonin(1A) receptor tagged to enhanced yellow fluorescent protein (5-HT(1A)R-EYFP) in live cells utilizing time-resolved fluorescence anisotropy decay. We interpret the unresolved fast component of the observed anisotropy decay as fluorescence resonance energy transfer (FRET) between 5-HT(1A)R-EYFP molecules (homo-FRET). Homo-FRET enjoys certain advantages over hetero-FRET in the analysis of receptor oligomerization. Our results reveal the presence of constitutive oligomers of the serotonin(1A) receptor in live cells. We further show that the oligomerization status of the receptor is independent of ligand stimulation and sphingolipid depletion. Interestingly, acute (but not chronic) cholesterol depletion appears to enhance the oligomerization process. Importantly, our results are independent of receptor expression level, thereby ruling out complications arising due to high expression. These results have potential implications in future therapeutic strategies in pathophysiological conditions in which serotonin(1A) receptors are implicated.

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.

Desmosterol replaces cholesterol for ligand binding function of the serotonin(1A) receptor in solubilized hippocampal membranes: support for nonannular binding sites for cholesterol?

The serotonin(1A) receptor is an important member of the G-protein coupled receptor family, and is involved in the generation and modulation of a variety of cognitive and behavioral functions. Solubilization of the hippocampal serotonin(1A) receptor by CHAPS is accompanied by loss of cholesterol that results in a reduction in specific agonist binding activity. Replenishment of cholesterol to solubilized membranes restores membrane cholesterol content and significantly recovers specific agonist binding. In order to test the stringency of cholesterol requirement, we solubilized native hippocampal membranes followed by replenishment with desmosterol. Desmosterol is the immediate biosynthetic precursor of cholesterol in the Bloch pathway differing only in a double bond at the 24th position. Our results show that replenishment with desmosterol restores ligand binding of serotonin(1A) receptors. This is consistent with earlier results showing that desmosterol can replace cholesterol in a large number of cases. However, these results appear to be contradictory to our earlier findings, performed by sterol manipulation utilizing methyl-ß-cyclodextrin, in which we observed that replacing cholesterol with desmosterol is unable to restore specific ligand binding of the hippocampal serotonin(1A) receptor. We discuss the possible molecular mechanism, in terms of nonannular lipid binding sites around the receptor, giving rise to these differences.

Cholesterol depletion enhances adrenergic signaling in cardiac myocytes.

Cardiac myocytes endogenously express α and ß adrenergic receptors, prototypes of the G-protein coupled receptor superfamily. Depending upon the dose of norepinephrine (agonist) exposure, hypertrophy and apoptosis are initiated by differential induction of two discrete constituents of the transcription factor AP-1, i.e., FosB and Fra-1. We explored differential adrenergic signaling as a paradigm for understanding how cholesterol dictates cells to choose hypertrophy or apoptosis. For this, we used fosB and fra-1 promoter-reporter constructs for monitoring adrenergic signaling. We show that cholesterol depletion enhances norepinephrine-mediated signaling in cardiac myocytes. Importantly, this increased signaling is reduced to original level upon cholesterol replenishment. We used specific ligands for α and ß adrenergic receptors and show that the enhanced signaling upon cholesterol depletion is a combined effect of both α and ß adrenergic receptors. These results constitute the first report demonstrating the effect of cholesterol on adrenergic signaling using a direct end-point gene expression.

Molecular modeling of the human serotonin(1A) receptor: role of membrane cholesterol in ligand binding of the receptor.

Serotonin(1A) receptors are important neurotransmitter receptors and belong to the superfamily of G-protein coupled receptors (GPCRs). Although it is an important drug target, the crystal structure of the serotonin(1A) receptor has not been solved yet. Earlier homology models of the serotonin(1A) receptor were generated using rhodopsin as a template. We have used two recent crystal structures of the human ß(2)-adrenergic receptor, one of which shows specific cholesterol binding site(s), as templates to model the human serotonin(1A) receptor. Since the sequence similarity between the serotonin(1A) receptor and ß(2)-adrenergic receptor is considerably higher than the similarity between the serotonin(1A) receptor and rhodopsin, our model is more reliable. Based on these templates, we generated models of the serotonin(1A) receptor in the absence and presence of cholesterol. The receptor model appears more compact in the presence of cholesterol. We validated the stability of 'compactness' using coarse-grain MD simulation. Importantly, all ligands exhibit higher binding energies when docked to the receptor in the presence of cholesterol, thereby implying that membrane cholesterol facilitates ligand binding to the serotonin(1A) receptor. To the best of our knowledge, this is one of the first reports in which lipid-specific receptor conformations have been modeled by homology modeling.

Amphotericin B inhibits entry of Leishmania donovani into primary macrophages.

Visceral leishmaniasis is a vector-borne disease caused by an obligate intra-macrophage protozoan parasite Leishmania donovani. The molecular mechanisms involved in internalization of Leishmania are still poorly understood. Amphotericin B and its formulations are considered as the best existing drugs against visceral leishmaniasis and are being increasingly used. The reason for its antileishmanial activity is believed to be its ability to bind ergosterol found in parasite membranes. In case of in vivo amphotericin B treatment, both host macrophages and parasites are exposed to amphotericin B. The effect of amphotericin B treatment could therefore be due to a combination of its interaction with both sterols i.e., ergosterol of Leishmania and cholesterol of host macrophages. We report here that cholesterol complexation by amphotericin B markedly inhibits binding of L. donovani promastigotes to macrophages. These results represent one of the first reports on the effect of amphotericin B on the binding of Leishmania parasites to host macrophages. Importantly, these results offer the possibility of reevaluating the mechanism behind the effectiveness of current therapeutic strategies that employ sterol-complexing agents such as amphotericin B to treat leishmaniasis.

Chronic cholesterol depletion using statin impairs the function and dynamics of human serotonin(1A) 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.

Membrane cholesterol in the function and organization of G-protein coupled receptors.

Cholesterol is an essential component of higher eukaryotic membranes and plays a crucial role in membrane organization, dynamics and function. The G-protein coupled receptors (GPCRs) are the largest class of molecules involved in signal transduction across membranes, and represent major targets in the development of novel drug candidates in all clinical areas. Membrane cholesterol has been reported to have a modulatory role in the function of a number of GPCRs. Two possible mechanisms have been previously suggested by which membrane cholesterol could influence the structure and function of GPCRs (i) through a direct/specific interaction with GPCRs, or (ii) through an indirect way by altering membrane physical properties in which the receptor is embedded, or due to a combination of both. Recently reported crystal structures of GPCRs have shown structural evidence of cholesterol binding sites. Against this backdrop, we recently proposed a novel mechanism by which membrane cholesterol could affect structure and function of GPCRs. According to our hypothesis, cholesterol binding sites in GPCRs could represent 'nonannular' binding sites. Interestingly, previous work from our laboratory has demonstrated that membrane cholesterol is required for the function of the serotonin(1A) receptor (a representative GPCR), which could be due to specific interaction of the receptor with cholesterol. Based on these results, we envisage that there could be specific/nonannular cholesterol binding site(s) in the serotonin(1A) receptor. We have analyzed putative cholesterol binding sites from protein databases in the serotonin(1A) receptor. Our analysis shows that cholesterol binding sites are inherent characteristic features of serotonin(1A) receptors and are conserved through natural evolution. Progress in deciphering molecular details of the GPCR-cholesterol interaction in the membrane would lead to better insight into our overall understanding of GPCR function in health and disease, thereby enhancing our ability to design better therapeutic strategies to combat diseases related to malfunctioning of GPCRs.

Metabolic depletion of sphingolipids impairs ligand binding and signaling of human serotonin1A receptors.

Sphingolipids are essential components of eukaryotic cell membranes and are thought to be involved in a variety of cellular functions. Mycotoxins such as fumonisins can disrupt sphingolipid metabolism, and treatment with fumonisins represents an efficient approach to modulate cellular sphingolipid levels. In this work, we modulated sphingolipid levels in CHO cells stably expressing the human serotonin(1A) receptor by metabolically inhibiting the biosynthesis of sphingolipids using fumonisin B(1). Serotonin(1A) receptors belong to the family of seven-transmembrane domain receptors that couple to G-proteins and are implicated in the generation and modulation of various cognitive, behavioral, and developmental functions. We explored the function of serotonin(1A) receptors under sphingolipid-depleted conditions by monitoring ligand binding, G-protein coupling, and downstream signaling of the receptor. Importantly, our results show that the function of the serotonin(1A) receptor is impaired upon metabolic depletion of sphingolipids, although the membrane receptor level does not exhibit any reduction. Interestingly, we find that the replenishment of sphingolipids using sphingosine results in restoration of ligand binding of serotonin(1A) receptors, demonstrating that the loss of ligand binding due to metabolic depletion of sphingolipids is reversible. These novel results demonstrate that sphingolipids are necessary for ligand binding and downstream signaling of the human serotonin(1A) receptor. We discuss possible mechanisms of specific interaction of sphingolipids with the serotonin(1A) receptor that could involve the proposed "sphingolipid-binding domain" (SBD).

Membrane cholesterol depletion enhances ligand binding function of human serotonin1A receptors in neuronal cells.

Membrane lipid composition of cells in the nervous system is unique and displays remarkable diversity. Cholesterol metabolism and homeostasis in the central nervous system and their role in neuronal function represent important determinants in neuropathogenesis. The serotonin(1A) receptor is an important member of the G-protein coupled receptor superfamily, and is involved in a variety of cognitive, behavioral, and developmental functions. We report here, for the first time, that the ligand binding function of human serotonin(1A) receptors exhibits an increase in membranes isolated from cholesterol-depleted neuronal cells. Our results gain pharmacological significance in view of the recently described structural evidence of specific cholesterol binding site(s) in GPCRs, and could be useful in designing better therapeutic strategies for neurodegenerative diseases associated with GPCRs.

Membrane cholesterol depletion from live cells enhances the function of human serotonin(1A) receptors.

Work from our laboratory has previously demonstrated the requirement of membrane cholesterol in the function of the serotonin(1A) receptor, a member of the G-protein coupled receptor (GPCR) superfamily. In order to monitor the effect of cellular organization on the function of human serotonin(1A) receptors, we explored receptor function following cholesterol depletion in live cells and membranes isolated from cholesterol-depleted cells. We report here the novel observation that while ligand binding of serotonin(1A) receptors displays an increase in membranes isolated from cholesterol-depleted cells, such trend is absent when binding is performed on cholesterol-depleted intact cells. Importantly, we show here, for the first time, that G-protein coupling of the serotonin(1A) receptor is enhanced in membranes isolated from cholesterol-depleted cells. These results assume pharmacological relevance in view of the recently described structural evidence of specific cholesterol binding sites in GPCRs, and may help in designing better therapeutic strategies for diseases related to GPCRs.

Differential effects of cholesterol and desmosterol on the ligand binding function of the hippocampal serotonin(1A) receptor: implications in desmosterolosis.

Cholesterol is a unique molecule in terms of high level of in-built stringency, fine tuned by natural evolution for its ability to optimize physical properties of higher eukaryotic cell membranes in relation to biological functions. We previously demonstrated the requirement of membrane cholesterol in maintaining the ligand binding activity of the hippocampal serotonin(1A) receptor. In order to test the molecular stringency of the requirement of cholesterol, we depleted cholesterol from native hippocampal membranes followed by replenishment with desmosterol. Desmosterol is an immediate biosynthetic precursor of cholesterol in the Bloch pathway differing only in a double bond at the 24th position in the alkyl side chain. Our results show that replenishment with desmosterol does not restore ligand binding activity of the serotonin(1A) receptor although replenishment with cholesterol led to significant recovery of ligand binding. This is in spite of similar membrane organization (order) in these membranes, as monitored by fluorescence anisotropy measurements. The requirement for restoration of ligand binding activity therefore appears to be more stringent than the requirement for the recovery of overall membrane order. These novel results have potential implications in understanding the interaction of membrane lipids with this important neuronal receptor in diseases such as desmosterolosis.

Are specific nonannular cholesterol binding sites present in G-protein coupled receptors?

The G-protein coupled receptors (GPCRs) are the largest class of molecules involved in signal transduction across membranes, and represent major drug targets in all clinical areas. Membrane cholesterol has been reported to have a modulatory role in the function of a number of GPCRs. Interestingly, recently reported crystal structures of GPCRs have shown structural evidence of cholesterol binding sites. Two possible mechanisms have been previously suggested by which membrane cholesterol could influence the structure and function of GPCRs (i) through a direct/specific interaction with GPCRs, which could induce a conformational change in the receptor, or (ii) through an indirect way by altering the membrane physical properties in which the receptor is embedded or due to a combination of both. We discuss here a novel mechanism by which membrane cholesterol could affect structure and function of GPCRs and propose that cholesterol binding sites in GPCRs could represent 'nonannular' binding sites. Interestingly, previous work from our laboratory has demonstrated that membrane cholesterol is required for the function of the serotonin1A receptor, which could be due to specific interaction of the receptor with cholesterol. Based on these results, we envisage that there could be specific/nonannular cholesterol binding site(s) in the serotonin1A receptor. We have analyzed putative cholesterol binding sites from protein databases in the serotonin1A receptor, a representative GPCR, for which we have previously demonstrated specific requirement of membrane cholesterol for receptor function. Our analysis shows that cholesterol binding sites are inherent characteristic features of serotonin1A receptors and are conserved over evolution. Progress in deciphering molecular details of the nature of GPCR-cholesterol interaction in the membrane would lead to better insight into our overall understanding of GPCR function in health and disease, thereby enhancing our ability to design better therapeutic strategies to combat diseases related to malfunctioning of GPCRs.

The function of G-protein coupled receptors and membrane cholesterol: specific or general interaction?

Cholesterol is an essential component of eukaryotic membranes and plays a crucial role in membrane organization, dynamics and function. The G-protein coupled receptors (GPCRs) are the largest class of molecules involved in signal transduction across membranes and constitute ~1-2% of the human genome. GPCRs have emerged as major targets for the development of novel drug candidates in all clinical areas due to their involvement in the generation of multitude of cellular responses. Membrane cholesterol has been reported to have a modulatory role in the function of a number of GPCRs. This effect could either be due to specific molecular interaction between cholesterol and GPCR, or due to alterations in the membrane physical properties induced by cholesterol. Alternatively, membrane cholesterol could modulate receptor function by occupying the 'nonannular' sites around the receptor. In this review, we have highlighted the nature of cholesterol dependence of GPCR function taking a few known examples.

Signaling by the human serotonin(1A) receptor is impaired in cellular model of Smith-Lemli-Opitz Syndrome.

The Smith-Lemli-Opitz Syndrome (SLOS) is a congenital and developmental malformation syndrome associated with defective cholesterol biosynthesis. SLOS is clinically diagnosed by reduced plasma levels of cholesterol along with elevated levels of 7-dehydrocholesterol (and its positional isomer 8-dehydrocholesterol) and the ratio of their concentrations to that of cholesterol. Since SLOS is associated with neurological deformities and malfunction, exploring the function of neuronal receptors and their interaction with membrane cholesterol under these conditions assumes significance. We have earlier shown the requirement of membrane cholesterol for the ligand binding function of an important neurotransmitter G-protein coupled receptor, the serotonin(1A) receptor. In the present work, we have generated a cellular model of SLOS using CHO cells stably expressing the human serotonin(1A) receptor. This was achieved by metabolically inhibiting the biosynthesis of cholesterol, utilizing a specific inhibitor (AY 9944) of the enzyme required in the final step of cholesterol biosynthesis. We utilized this cellular model to monitor the function of the human serotonin(1A) receptor under SLOS-like condition. Our results show that ligand binding activity, G-protein coupling and downstream signaling of serotonin(1A) receptors are impaired in SLOS-like condition, although the membrane receptor level does not exhibit any reduction. Importantly, metabolic replenishment of cholesterol using serum partially restored the ligand binding activity of the serotonin(1A) receptor. These results are potentially useful in developing strategies for the future treatment of the disease since intake of dietary cholesterol is the only feasible treatment for SLOS patients.

Differential effects of cholesterol and its immediate biosynthetic precursors on membrane organization.

Cholesterol is the most representative sterol present in vertebrate membranes and is the end product of the long and multistep sterol biosynthetic pathway. 7-Dehydrocholesterol (7-DHC) and desmosterol are the immediate biosynthetic precursors of cholesterol in the Kandutsch-Russell and Bloch pathway. In this article, we have monitored the effect of cholesterol and its two immediate biosynthetic precursors on biophysical and dynamic properties of fluid and gel phase membranes. Toward this goal, we have used fluorescent membrane probes, DPH and TMA-DPH, and the hydrophobic probe, pyrene. Our results using these probes show that although both 7-DHC and desmosterol differ with cholesterol in one double bond, they exhibit differential effects on membrane organization and dynamics. Importantly, we show that the effect of cholesterol and desmosterol on membrane organization and dynamics is similar in most cases, while 7-DHC has a considerably different effect. This demonstrates that the position of the double bond in sterols is an important determinant in maintaining membrane order and dynamics. These results assume relevance since the accumulation of cholesterol precursors have been reported to result in severe pathological conditions.