The role of membrane cholesterol as a crucial regulator in the structure and function of membrane proteins and receptors is well documented. However, there is a lack of consensus on the mechanism for such regulation. We have previously shown that the function of an important neuronal receptor, the serotonin1A receptor, is modulated by cholesterol in hippocampal membranes. With an overall objective of addressing the role of 3 membrane physical properties in receptor function, we measured the viscosity of hippocampal membranes of varying cholesterol content using a meso-substituted fluorophore (BODIPY-C12) based on the BODIPY probe. BODIPY-C12 acts as a fluorescent molecular rotor and allows measurement of hippocampal membrane viscosity through its characteristic viscosity-sensitive fluorescence depolarization. A striking feature of our results is that specific agonist binding by the serotonin1A receptor exhibits close correlation with hippocampal membrane viscosity, implying the importance of global membrane properties in receptor function. We envision that our results are important in understanding GPCR regulation by the membrane environment, and is relevant in the context of diseases in which GPCR signaling plays a major role and are characterized by altered membrane fluidity.Abbreviations: 2-AS, 2-(9-anthroyloxy)stearic acid; 12-AS, 12-(9-anthroyloxy)stearic acid; BODIPY, 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; GPCR, G protein-coupled receptor; 8-12-PC, 1-palmitoyl-2-(12-doxyl)stearoyl-sn-glycero-3-phosphocholine; DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine; DOPC, 1,2-dioleoylsn-glycero-3-phosphocholine; MβCD, methyl-β-cyclodextrin; Tempo-PC, 1,2-dioleoyl-snglycero-3-phosphotempocholine Keywords: Molecular rotor; hippocampal membrane; viscosity; cholesterol; neuronal receptor IntroductionBiological membranes are complex, highly organized, two-dimensional, supramolecular assemblies of a diverse variety of lipids and proteins. The function of membranes is to allow cellular compartmentalization, and impart an identity to individual cells and organelles, besides providing an appropriate environment for proper functioning of membrane proteins. Interestingly, cellular membranes in the nervous system are characterized by very high concentration and remarkable diversity of lipids, and these are correlated with increased complexity in the function of the nervous system (Sastry, 1985;Wenk, 2005). In this context, cholesterol represents an important lipid since brain cholesterol has been implicated in a number of neurological disorders (Chattopadhyay and Paila, 2007;Martín et al., 2014), some of which share a common etiology of defective cholesterol 4 metabolism in the brain (Porter and Herman, 2011). More importantly, the function of neuronal receptors depends on cholesterol (Pucadyil and Chattopadhyay, 2006;Allen et al., 2007;Paila and Chattopadhyay, 2010;Jafurulla and Chattopadhyay, 2013), which affects neurotransmission, resulting in mood and anxiety disorders (Papakostas et al., 2004). In spite of ...

Biological membranes are highly organized supramolecular assemblies of lipids and proteins. The membrane interface separates the outer (bulk) aqueous phase from the hydrophobic membrane interior. In this work, we have explored the microstructure and collective dynamics of the membrane interfacial hydration shell in zwitterionic and negatively charged phospholipid membrane bilayers using terahertz time-domain spectroscopy. We show here that the relaxation time constants of the water hydrogen bond network exhibit a unique "rise and dip" pattern with increasing lipid concentration. More importantly, we observed a dependence of the critical lipid concentration corresponding to the inflection point on the charge of the lipid headgroup, thereby implicating membrane electrostatics as a major factor in the microstructure and dynamics of water at the membrane interface. These results constitute one of the first experimental evidences of the modulation of the dielectric relaxation response of membrane interfacial water by membrane lipid composition in a concentration-dependent manner. Lipid-stringent membrane hydration could be relevant in the broader context of lipid diversity observed in biological membranes and the role of negatively charged lipids in membrane protein structure and function.

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Water drives the spontaneous self-assembly of lipids and proteins into quasi two-dimensional biological membranes that act as catalytic scaffolds for numerous processes central to life. However, the functional relevance of hydration in membrane biology is only beginning to be addressed, predominantly because of challenges associated with direct measurements of hydration microstructure and dynamics in a biological milieu. Our recent work on the novel interplay of membrane electrostatics and crowding in shaping membrane hydration dynamics utilizing terahertz (THz) spectroscopy represents an important step in this context. In this Perspective, we provide a glimpse into the ever-broadening functional landscape of hydration dynamics in biological membranes in the backdrop of the unique physical chemistry of water molecules. We further highlight the immense (and largely untapped) potential of the THz toolbox in addressing contemporary problems in membrane biology, while emphasizing the adaptability of the analytical framework reported recently by us to such studies.

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Understanding the mechanism of action of local anesthetics has been challenging. We previously showed that the local anesthetic phenylethanol (PEtOH) inhibits the function of serotonin1A receptor, which is a member of the G protein-coupled receptor family and a neurotransmitter receptor. With the objective of gaining insight into the molecular mechanism underlying the anesthetic (PEtOH) action, we monitored the organization and dynamics of hippocampal membranes using multiple fluorescent reporters, which include a molecular rotor (BODIPY-C12) and a voltage-sensitive probe (4-(2-(6-(dioctylamino)-2-naphthalenyl)­ethenyl)-1-(3-sulfopropyl)-pyridinium inner salt) (di-8-ANEPPS), besides pyrene. These interfacial membrane probes were chosen because membrane partitioning of PEtOH would be reflected in the membrane interfacial environment. Taken together, we report a reduction in dipole potential and microviscosity of hippocampal membranes, with a concomitant increase in lateral diffusion in the presence of PEtOH. The reduction in membrane dipole potential induced by PEtOH constitutes one of the first experimental demonstrations on the modulation of membrane dipole potential by local anesthetics. Our results assume significance in view of previous reports that correlate membrane-perturbing effects of local anesthetics to their anesthetic action. We envision that insights into the interaction of local anesthetics with membranes could serve as a crucial link in developing a comprehensive understanding of the molecular mechanisms involved in anesthesia.