Translocating sensors

Translocating Sensors in Living Cells A second approach for monitoring small molecules with genetically encoded sensors is the use of translocation properties of proteins or protein domains inside cells [105]. One advantage compared to HIET sensors is the fact that only a single fusion of the translocating protein with a fluorophore, mostly a fluorescent protein, is required. Many proteins... 

In primary cultures of neonatal cerebellar granule neurons, all Ca2+ sensors, calmodulin, protein kinases C (PKC), and the p21(ras)/phosphatidylinositol 3 -kinase (Ptdlns-3K)/Akt pathway, converge towards NF-kB at the levels of nuclear translocation as well as transcription. The duration of NF-kB activation is a critical determinant for sensitivity toward excitotoxic stress and is dependent on the different upstream and downstream signaling associated with various kinases. This is in contrast to studies in non-neuronal cells, which either do not respond to Ca2+ or do not simultaneously activate all three cascades (Lilienbaum and Israel, 2003). Collective evidence suggests that brain inflammatory processes differ from systemic inflammation not only in the involvement of various types of neural cells but also in differences in response to second messengers. 

Fig. 6.3 Steady state relation between the metabolite S normalized by the value, Si, = vmaxGKf/b and the glucose concentration, G, for different values of the maximal active fraction Xmax. The smallest active fraction value, Xq, is set equal to 0.1. The main effect of the translocation of glucokinase is a right hand shift of the relation, resulting in a threshold value forthe glucose sensor.

The main variation in the uptake is therefore taking place by varying the amount of active GLUT4 at the cell membrane [59-61, 82]. This is one of the important actions of insulin, but neural activity can also translocate GLUT4 independently of insulin [85]. This probably takes place during exercise [61, 82] and during stimulation from the portal sensor [44]. The glucose influx over the cell membrane can then be described like ... 

Especially at the beginning it is important that the nervous system is able to sense the meal and start the GEUT4 translocation. Here the activity of the hepato-portal sensor is ideal, because it gives a signal that is precisely proportional to Jabs. It is typically seen that the plasma insulin concentration increases before any noticeable increase in glucose concentration [121],... 

A schematic representation of a FRET-based voltage sensor assay is shown in Fig. 13. The assay principle was first published [105] and then further improved [106] by Gonzalez and Tsien, then commercialized [107], and is now available from Panvera [108]. The FRET donor is a coumarin dye, which is covalently linked to a phosphoHpid. The acceptor is a highly fluorescent, membrane-soluble anionic ox-onol dye. When the cell membrane is loaded with the dyes, the phospholipid anchors the coumarin donor to the outside of the cell, whereas the oxonol dye is accumulated in the ceU membrane. The distribution of the anionic oxonol in the membrane depends on the polarity of the membrane potential if the oxonol dye is located on the extracellular side of the membrane in close proximity to the coumarin donor, FRET occurs and the emission is mostly at 580 nm. If the polarity changes, the oxonol rapidly translocates to the intracellular side of the membrane, too far from the coumarin donor for FRET, and the emission is mostly at 460 nm. 

The PAS domain transcriptional factor AhR is a nonnuclear receptor xenobiotic receptor. In the early 1990s, the AhR and its partner Ah receptor nuclear translocator (Arnt) protein were identified as a transcriptional sensor mediating the induction of CYP1A and 1B1 genes by dioxin and related polycyclic aromatic hydrocarbons [62, 63],... 

During the last decade numerous applications were developed employing fluorescent proteins. This includes passive applications such as the use of fluorescent proteins as fluorescence tags in fusion proteins to monitor the appearance, degradation, location or translocation of appropriate partner proteins, as well as more active applications measuring biochemical parameters such as metabolite concentrations, enzyme activity, or protein-protein interactions by their effects on the fluorescence properties of appropriately designed derivatives of fluorescent proteins (biochemical sensors/indicators) [76]. 

Micromolar amounts of aluminum hydroxide profoundly reduce the voltage dependence of VDAC (36, 37). Although initial results were consistent with a direct neutralization of the voltage sensor (36), further work (30) indicates that an indirect effect is more likely. The presence of aluminum hydroxide in the compartment on one side of a membrane inhibits channel closure when that side is made negative. Positive potentials on the aluminum side result in VDAC closure, but channel reopening is inhibited. This phenomenon can be explained in terms of an aluminum hydroxide binding site that is translocated across the membrane (30). 

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