Studying ion channel conformation dynamics by encoding coumarin as unnatural amino acid.

Monitoring the conformational changes of proteins is critical to understand their function. Ion channels are membrane-bound minute machines controlling the passage of ions across biological membranes. The precise labeling of ion channels with fluorescent probes allows studying their dynamics and facilitates their characterization by high-resolution optical techniques. Here we describe a protocol for the use of a small fluorescent reporter, incorporated by expansion of the genetic code in the host cell. An important advantage of using small probes is that they are less likely to perturb protein structure, function, and trafficking. In our hands, Tyr-coumarin proved to be useful to measure the conformational changes occurring in the narrow space of the permeation pathway in single capsaicin receptors. The method described here could be directly translated to the study of membrane receptors, non-electrogenic transporters, or membrane-bound enzymes.

A rationally designed orthogonal synthetase for genetically encoded fluorescent amino acids.

The incorporation of non-canonical amino acids into proteins has emerged as a promising strategy to manipulate and study protein structure-function relationships with superior precision in vitro and in vivo. To date, fluorescent non-canonical amino acids (f-ncAA) have been successfully incorporated in proteins expressed in bacterial systems, Xenopus oocytes, and HEK-293T cells. Here, we describe the rational generation of a novel orthogonal aminoacyl-tRNA synthetase based on the E. coli tyrosine synthetase that is capable of encoding the f-ncAA tyr-coumarin in HEK-293T cells.

Glucose Increase DAGLα Levels in Tanycytes and Its Inhibition Alters Orexigenic and Anorexigenic Neuropeptides Expression in Response to Glucose.

The endocannabinoid system (ECS) is composed of a group of Gi-coupled protein receptors and enzymes, producing and degrading the endocannabinoids, 2-arachidonoylglycerol (2-AG) and N-arachidonoyl-ethanolamine (AEA). Endocannabinoid-mediated signaling modulates brain functions, such as pain, mood, memory, and feeding behavior. The activation of the ECS is associated with overeating and obesity; however, the expression of components of this system has been only partially studied in the hypothalamus, a critical region implicated in feeding behavior. Within this brain region, anorexigenic, and orexigenic neurons of the arcuate nucleus (ARC) are in close contact with tanycytes, glial radial-like cells that line the lateral walls and floor of the third ventricle (3V). The specific function of tanycytes and the effects of metabolic signals generated by them on adjacent neurons is starting to be elucidated. We have proposed that the ECS within tanycytes modulates ARC neurons, thus modifying food intake. Here, we evaluated the expression and the loss of function of the 2-AG-producing enzyme, diacylglycerol lipase-alpha (DAGLα). Using Western blot and immunohistochemistry analyses in basal hypothalamus sections of adult rats under several glycemic conditions, we confirm that DAGLα is strongly expressed at the basal hypothalamus in glial and neuronal cells, increasing further in response to greater extracellular glucose levels. Using a DAGLα-inhibiting adenovirus (shRNA), suppression of DAGLα expression in tanycytes altered the usual response to intracerebroventricular glucose in terms of neuropeptides produced by neurons of the ARC. Thus, these results strongly suggest that the tanycytes could generate 2-AG, which modulates the function of anorexigenic and orexigenic neurons.

Conformational dynamics in TRPV1 channels reported by an encoded coumarin amino acid.

TRPV1 channels support the detection of noxious and nociceptive input. Currently available functional and structural data suggest that TRPV1 channels have two gates within their permeation pathway: one formed by a 'bundle-crossing' at the intracellular entrance and a second constriction at the selectivity filter. To describe conformational changes associated with channel gating, the fluorescent non-canonical amino acid coumarin-tyrosine was genetically encoded at Y671, a residue proximal to the selectivity filter. Total internal reflection fluorescence microscopy was performed to image the conformational dynamics of the channels in live cells. Photon counts and optical fluctuations from coumarin encoded within TRPV1 tetramers correlates with channel activation by capsaicin, providing an optical marker of conformational dynamics at the selectivity filter. In agreement with the fluorescence data, molecular dynamics simulations display alternating solvent exposure of Y671 in the closed and open states. Overall, the data point to a dynamic selectivity filter that may serve as a gate for permeation.

Cells use proteins on their surface as sensors to help them to assess and explore their environments and adapt to external conditions. The transient receptor potential (TRP) ion channels form one such family of proteins. Sodium, potassium and calcium ions can move through TRP channels to enter and exit cells, and by doing so trigger changes in the cell that help it respond to an external stimulus. The channels have physical "gates" that can open and close to control the flow of the ions. When the TRP channel detects a stimulus ­ which could take the form of specific chemicals, or a change in temperature, pressure or voltage ­ it changes shape, causing the gate to open. Researchers have a number of unanswered questions about how TRP channels work. Where in the channels are gates located? How do the channels control the flow of ions, and how do they communicate with each other? And which regions of the protein sense environmental cues? As a result, new technologies are being developed to make it easier to study the types of rearrangements that TRP channels experience when they activate. Steinberg, Kasimova et al. have used total internal reflection microscopy ­ a method that images fluorescent molecules ­ to measure the conformational change of a single TRP channel in a living cell. This channel, called TRPV1, senses external heat and plays an important role in pain perception. Its gate can also be opened by the pungent compound of chili pepper, capsaicin. The results of the experiments suggest that a selectivity filter region in TRPV1 channels changes its shape when the channel opens in response to capsaicin. Then, this selectivity filter appears to do double duty ­ it controls which types of ions pass through the channels as well as controlling their flow rate. Because of its role in pain perception, having a better understanding of how TRPV1 works will be valuable for researchers who are trying to develop new pain relief treatments. The so-called 'seeing is believing' method used by Steinberg, Kasimova et al. could also be used to study other membrane proteins, both to guide drug development and to improve our understanding of how cells interact with their environment.

The Different Roles of The Channel-Kinases TRPM6 and TRPM7.

Melastatin-related Transient Receptor Potential 6 and 7 (TRPM6 and TRPM7) are cation channels with the almost unique trait of each possessing a kinase domain in its C terminus. Both the transmembrane pore and kinase are functional, and have been characterized experimentally, but whether one domain regulates the function of the other, or vice versa has remained largely unsettled. These proteins play important physiological roles in magnesium homeostasis and other cellular processes such as cell death, proliferation, differentiation and migration, and are consequently associated with several types of pathologies. Recently, studies performed in mice expressing a TRPM7 kinase-dead mutant suggest that the enzyme may function as part of a Mg(2+) sensor and transducer of signaling pathways during stressful environmental conditions. Additionally, it has been shown that TRPM7's kinase can act on its own in chromatin remodeling processes. Thus, the recent work in this field has provided new insights into the function of these interesting proteins and how they might be involved in human disease.

A structural view of ligand-dependent activation in thermoTRP channels.

Transient Receptor Potential (TRP) proteins are a large family of ion channels, grouped into seven sub-families. Although great advances have been made regarding the activation and modulation of TRP channel activity, detailed molecular mechanisms governing TRP channel gating are still needed. Sensitive to electric, chemical, mechanical, and thermal cues, TRP channels are tightly associated with the detection and integration of sensory input, emerging as a model to study the polymodal activation of ion channel proteins. Among TRP channels, the temperature-activated kind constitute a subgroup by itself, formed by Vanilloid receptors 1-4, Melastatin receptors 2, 4, 5, and 8, TRPC5, and TRPA1. Some of the so-called "thermoTRP" channels participate in the detection of noxious stimuli making them an interesting pharmacological target for the treatment of pain. However, the poor specificity of the compounds available in the market represents an important obstacle to overcome. Understanding the molecular mechanics underlying ligand-dependent modulation of TRP channels may help with the rational design of novel synthetic analgesics. The present review focuses on the structural basis of ligand-dependent activation of TRPV1 and TRPM8 channels. Special attention is drawn to the dissection of ligand-binding sites within TRPV1, PIP2-dependent modulation of TRP channels, and the structure of natural and synthetic ligands.

Human CCAAT/enhancer-binding protein ß interacts with chromatin remodeling complexes of the imitation switch subfamily.

Transcription factor C/EBPß is involved in several cellular processes, such as proliferation, differentiation, and energy metabolism. This factor exerts its activity through recruitment of different proteins or protein complexes, including the ATP-dependent chromatin remodeling complex SWI/SNF. The C/EBPß protein is found as three major isoforms, C/EBPß1, -2, and -3. They are generated by translation at alternative AUG initiation codons of a unique mRNA, C/EBPß1 being the full-length isoform. It has been found that C/EBPß1 participates in terminal differentiation processes. Conversely, C/EBPß2 and -3 promote cell proliferation and are involved in malignant progression in a number of tissues. The mechanisms by which C/EBPß2 and -3 promote cell proliferation and tumor progression are not fully understood. In this work, we sought to identify proteins interacting with hC/EBPß using a proteomics approach. We found that all three isoforms interact with hSNF2H and hACF, components of ACF and CHRAC chromatin remodeling complexes, which belong to the imitation switch subfamily. Additional protein-protein interaction studies confirmed this finding and also showed that hC/EBPß directly interacts with hACF1. By overexpressing hC/EBPß, hSNF2H, and hACF1 in HepG2 cells and analyzing variations in expression of cyclin D1 and other C/EBPß target genes, we observed a functional interaction between C/EBPß and SNF2H/ACF1, characterized mainly by suppression of C/EBPß transactivation activity in the presence of SNF2H and ACF1. Consistent with these findings, induction of differentiation of HepG2 cells by 1% DMSO was accompanied by a reduction in the level of cyclin D1 expression and the appearance of hC/EBPß, hSNF2H, and hACF1 on the promoter region of this gene.