Shanti Kalipatnapu

The Serotonin1A Receptor: A Representative Member of the Serotonin Receptor Family

Abstract1. Serotonin is an intrinsically fluorescent biogenic amine that acts as a neurotransmitter and is found in a wide variety of sites in the central and peripheral nervous system. Serotonergic signaling appears to play a key role in the generation and modulation of various cognitive and behavioral functions.2. Serotonin exerts its diverse actions by binding to distinct cell surface receptors which have been classified into many groups. The serotonin1A (5-HT1A) receptor is the most extensively studied of the serotonin receptors and belongs to the large family of seven transmembrane domain G-protein coupled receptors.3. The tissue and sub-cellular distribution, structural characteristics, signaling of the serotonin1A receptor and its interaction with G-proteins are discussed.4. The pharmacology of serotonin1A receptors is reviewed in terms of binding of agonists and antagonists and sensitivity of their binding to guanine nucleotides.5. Membrane biology of 5-HT1A receptors is presented using the bovine hippocampal serotonin1A receptor as a model system. The ligand binding activity and G-protein coupling of the receptor is modulated by membrane cholesterol thereby indicating the requirement of cholesterol in maintaining the receptor organization and function. This, along with the reported detergent resistance characteristics of the receptor, raises important questions on the role of membrane lipids and domains in the function of this receptor.

Membrane Protein Solubilization: Recent Advances and Challenges in Solubilization of Serotonin1A Receptors

Solubilization of integral membrane proteins is a process in which the proteins and lipids that are held together in native membranes are suitably dissociated in a buffered detergent solution. The controlled dissociation of the membrane results in formation of small protein and lipid clusters that remain dissolved in the aqueous solution. Effective solubilization and purification of membrane proteins, especially heterologously‐expressed proteins in mammalian cells in culture, in functionally active forms represent important steps in understanding structure‐function relationship of membrane proteins. In this review, critical factors determining functional solubilization of membrane proteins are highlighted with the solubilization of the serotonin1A receptor taken as a specific example. IUBMB Life, 57: 505‐512, 2005

Solubilization of high affinity G-protein-coupled serotonin 1A receptors from bovine hippocampus using pre-micellar CHAPS at low concentration

The serotonin 1A (5-HT 1A ) receptors are members of a superfamily of seven transmembrane domain receptors that couple to G-proteins. They appear to be involved in various behavioural and cognitive functions. This paper reports an efficient strategy to solubilize 5-HT 1A receptors from bovine hippocampal membranes using the zwitterionic detergent CHAPS which is mild and non-denaturing. Since high concentration of CHAPS has earlier been shown to induce dissociation and depletion of G-protein sub-units, a low (pre-micellar) concentration of CHAPS was used for solubilizing 5-HT 1A receptors in the presence of NaCl followed by PEG precipitation. This results in solubilization of 5-HT 1A receptors with a high degree of efficiency and gives rise to high affinity, functionally active G-protein-sensitive solubilized receptors. Optimal solubilization of the receptor from the native source with high ligand binding affinity and intact signal transduction components may constitute the first step in the molecular characterization of the 5-HT 1A receptor in particular, and G-protein-coupled receptors in general.

Membrane Organization and Function of the Serotonin1A Receptor

Abstract(1) The serotonin1A receptor is a G-protein coupled receptor involved in several cognitive, behavioral, and developmental functions. It binds the neurotransmitter serotonin and signals across the membrane through its interactions with heterotrimeric G-proteins. (2) Lipid–protein interactions in membranes play an important role in the assembly, stability, and function of membrane proteins. The role of membrane environment in serotonin1A receptor function is beginning to be addressed by exploring the consequences of lipid manipulations on the ligand binding and G-protein coupling of serotonin1A receptors, the ability to functionally solubilize the serotonin1A receptor, and the factors influencing the membrane organization of the serotonin1A receptor. (3) Recent developments involving the application of detergent-based and detergent-free approaches to understand the membrane organization of the serotonin1A receptor under conditions of ligand activation and modulation of membrane lipid content, with an emphasis on membrane cholesterol, are described.

A GFP fluorescence-based approach to determine detergent insolubility of the human serotonin1A receptor.

Insolubility in non‐ionic detergents such as Triton X‐100 is a widely used biochemical criterion for characterization of membrane domains. We report here a novel green fluorescent protein fluorescence‐based approach to directly determine detergent insolubility of specific membrane proteins. We have applied this method to explore the detergent resistance of an important G‐protein coupled receptor, the serotonin1A (5‐HT1A) receptor. Our results show, for the first time, that a small yet significant fraction of the 5‐HT1A receptor exhibits detergent insolubility. These results are validated by control experiments involving fluorescent lipid probes and protein markers. Our results assume relevance in the context of localization of the 5‐HT1A receptor in membrane domains and its significance in receptor function and signaling.

Membrane organization of the human serotonin1A receptor monitored by detergent insolubility using GFP fluorescence

Insolubility in non-ionic detergents such as Triton X-100 at low temperature is a widely used biochemical criterion for characterization of membrane domains. In view of the emerging role of membrane organization in the function of G-protein coupled receptors, we have examined detergent insolubility of the 5-HT1A receptor in CHO cells using a novel GFP fluorescence approach developed by us. Using this approach, we have explored the membrane organization of the serotonin1A receptor tagged to enhanced yellow fluorescent protein (5-HT1AR-EYFP) stably expressed in CHO-K1 cells under conditions of varying detergent concentration, reduced membrane cholesterol and agonist stimulation. Our results show that a small yet significant fraction of the 5-HT1A receptor exhibits detergent insolubility, which increases upon depletion of membrane cholesterol. Stimulation of 5-HT1AR-EYFP by its endogenous ligand, serotonin, did not cause a significant change in the detergent insolubility of the receptor. Taken together, our results on detergent insolubility of 5-HT1AR-EYFP provide new insights into the membrane organization of the 5-HT1A receptor and could be relevant in the analysis of membrane organization of other G-protein coupled receptors.

Solubilization of Serotonin1A Receptors Heterologously Expressed in Chinese Hamster Ovary Cells

Abstract1. The serotonin1A (5-HT1A) receptors are members of a superfamily of seven transmembrane domain receptors that couple to G-proteins. They appear to be involved in various behavioral and cognitive functions.2. We report here, for the first time, the solubilization of 5-HT1A receptors stably expressed in Chinese Hamster Ovary (CHO) cells using the zwitterionic detergent CHAPS in presence of NaCl followed by polyethylene glycol (PEG) precipitation. We show by ligand-binding assay that the 5-HT1A receptor solubilized this way is functionally active. We have optimized the efficiency of solubilization with respect to total protein and NaCl concentration.3.Our results show that careful control of salt and protein concentration is crucial in optimal solubilization of membrane receptors heterologously expressed in cells in culture. The effective solubilization of important neurotransmitter receptors such as 5-HT1A receptors which are present in very low amounts in the native tissue may represent an important step in characterizing membrane receptors expressed in mammalian cells in culture.

Interaction of Serotonin1A Receptors From Bovine Hippocampus With Tertiary Amine Local Anesthetics

Abstract1. We have examined the interaction of tertiary amine local anesthetics with the bovine hippocampal serotonin1A (5-HT1A) receptor, an important member of the G-protein- coupled receptor superfamily.2. The local anesthetics inhibit specific agonist and antagonist binding to the 5-HT1A receptor at a clinically relevant concentration range of the anesthetics. This is accompanied by a concomitant reduction in the binding affinity of the 5-HT1A receptor to the agonist. Interestingly, the extent of G-protein coupling of the receptor is reduced in the presence of the local anesthetics.3. Fluorescence polarization measurements using depth-dependent fluorescent probes show that procaine and lidocaine do not show any significant change in membrane fluidity. On the other hand, tetracaine and dibucaine were found to alter fluidity of the membrane as indicated by a fluorescent probe which monitors the headgroup region of the membrane.4. The local anesthetics showed inhibition of agonist binding to the 5-HT1A receptor in membranes depleted of cholesterol more or less to the same extent as that of control membranes in all cases. This suggests that the inhibition in ligand binding to the 5-HT1A receptor brought about by local anesthetics is independent of the membrane cholesterol content.5. Our results on the effects of the local anesthetics on the ligand binding and G-protein coupling of the 5-HT1A receptor support the possibility that G-protein-coupled receptors could be involved in the action of local anesthetics.

Monitoring the organization and dynamics of bovine hippocampal membranes utilizing differentially localized fluorescent membrane probes.

Previous work from our laboratory has established bovine hippocampal membranes as a convenient natural source for studying neuronal receptors such as the G-protein coupled serotonin1A receptor. In this paper, we have explored the organization and dynamics of bovine hippocampal membranes using environment-sensitive and differentially localized fluorescent probes NBD-PE and NBD-cholesterol, utilizing wavelength-selective and time-resolved fluorescence measurements. The NBD group in NBD-PE is localized at the membrane interface while in NBD-cholesterol it is localized deeper in the membrane. Our results show that native hippocampal membranes offer considerable motional restriction as evidenced from red edge excitation shift of NBD probes. However, this effect progressively decreases with increasing cholesterol depletion in the case of NBD-cholesterol, possibly indicating a reduction in membrane heterogeneity. In contrast, REES of NBD-PE in hippocampal membranes does not show any significant change upon cholesterol depletion indicating relative lack of sensitivity of the membrane interface to cholesterol depletion. These observations are supported by changes in fluorescence polarization with cholesterol depletion. Taken together, these results imply that the deeper hydrocarbon region of the hippocampal membrane is more sensitive to changes in membrane organization and dynamics due to cholesterol depletion than the interfacial region. The motional restriction in native membranes is maintained even in the absence of proteins. The fluorescence lifetimes of both the NBD probes show slight reduction upon cholesterol depletion indicating a change in micro-environmental polarity possibly due to water penetration. These results are relevant in understanding the complex organization of hippocampal membranes and could have possible functional implications.

Membrane organization and dynamics of the G-protein-coupled serotonin1A receptor monitored using fluorescence-based approaches.

The G-protein-coupled receptor (GPCR) superfamily represents one of the largest classes of molecules involved in signal transduction across the plasma membrane. Fluorescence-based approaches have provided valuable insights into GPCR functions such as receptor–receptor and receptor–ligand interactions, real-time assessment of signal transduction, receptor dynamics on the plasma membrane, and intracellular trafficking of receptors. This has largely been possible with the use of fluorescent probes such as the green fluorescent protein (GFP) from the jellyfish Aequoria victoria and its variants. We discuss the potential of fluorescence-based approaches in providing novel information on the membrane organization and dynamics of the G-protein-coupled serotonin1A receptor tagged to the enhanced yellow fluorescent protein (EYFP).