Thomas Gensch

Chloride Accumulation in Mammalian Olfactory Sensory Neurons

The generation of an excitatory receptor current in mammalian olfactory sensory neurons (OSNs) involves the sequential activation of two distinct types of ion channels: cAMP-gated Ca2+-permeable cation channels and Ca2+-gated Cl- channels, which conduct a depolarizing Cl- efflux. This unusual transduction mechanism requires an outward-directed driving force for Cl-, established by active accumulation of Cl- within the lumen of the sensory cilia. We used two-photon fluorescence lifetime imaging microscopy of the Cl--sensitive dye 6-methoxy-quinolyl acetoethyl ester to measure the intracellular Cl- concentration in dendritic knobs of OSNs from mice and rats. We found a uniform intracellular Cl- concentration in the range of 40-50 mm, which is indicative of active Cl- accumulation. Functional assays and PCR experiments revealed that NKCC1-mediated Cl- uptake through the apical membrane counteracts Cl- depletion in the sensory cilia, and thus maintains the responsiveness of OSNs to odor stimulation. To permit Cl- accumulation, OSNs avoid the “chloride switch”: they do not express KCC2, the main Cl- extrusion cotransporter operating in neurons of the adult CNS. Cl- accumulation provides OSNs with the driving force for the depolarizing Cl- current that is the basis of the low-noise receptor current in these neurons.

Fluorescence Detection from Single Dendrimers with Multiple Chromophores

The differences in the fluorescence behavior of a polyphenylene dendrimer with eight peryleneimides chromophores (1) and a single hexaphenylperyleneimide chromophore have been investigated at a single-molecule level through the combination of ultrasensitive fluorescence detection and microscopy.

Real-time determination of intracellular oxygen in bacteria using a genetically encoded FRET-based biosensor.

BackgroundMolecular oxygen (O2) is one of the key metabolites of all obligate and facultative aerobic pro- and eukaryotes. It plays a fundamental role in energy homeostasis whereas oxygen deprivation, in turn, broadly affects various physiological and pathophysiological processes. Therefore, real-time monitoring of cellular oxygen levels is basically a prerequisite for the analysis of hypoxia-induced processes in living cells and tissues.ResultsWe developed a genetically encoded Förster resonance energy transfer (FRET)-based biosensor allowing the observation of changing molecular oxygen concentrations inside living cells. This biosensor named FluBO (fluorescent protein-based biosensor for oxygen) consists of the yellow fluorescent protein (YFP) that is sensitive towards oxygen depletion and the hypoxia-tolerant flavin-binding fluorescent protein (FbFP). Since O2 is essential for the formation of the YFP chromophore, efficient FRET from the FbFP donor domain to the YFP acceptor domain only occurs in the presence but not in the absence of oxygen. The oxygen biosensor was used for continuous real-time monitoring of temporal changes of O2 levels in the cytoplasm of Escherichia coli cells during batch cultivation.ConclusionsFluBO represents a unique FRET-based oxygen biosensor which allows the non-invasive ratiometric readout of cellular oxygen. Thus, FluBO can serve as a novel and powerful probe for investigating the occurrence of hypoxia and its effects on a variety of (patho)physiological processes in living cells.

Modulation of chloride homeostasis by inflammatory mediators in dorsal root ganglion neurons

BackgroundChloride currents in peripheral nociceptive neurons have been implicated in the generation of afferent nociceptive signals, as Cl- accumulation in sensory endings establishes the driving force for depolarizing, and even excitatory, Cl- currents. The intracellular Cl- concentration can, however, vary considerably between individual DRG neurons. This raises the question, whether the contribution of Cl- currents to signal generation differs between individual afferent neurons, and whether the specific Cl- levels in these neurons are subject to modulation. Based on the hypothesis that modulation of the peripheral Cl- homeostasis is involved in the generation of inflammatory hyperalgesia, we examined the effects of inflammatory mediators on intracellular Cl- concentrations and on the expression levels of Cl- transporters in rat DRG neurons.ResultsWe developed an in vitro assay for testing how inflammatory mediators influence Cl- concentration and the expression of Cl- transporters. Intact DRGs were treated with 100 ng/ml NGF, 1.8 μM ATP, 0.9 μM bradykinin, and 1.4 μM PGE2 for 1–3 hours. Two-photon fluorescence lifetime imaging with the Cl--sensitive dye MQAE revealed an increase of the intracellular Cl- concentration within 2 hours of treatment. This effect coincided with enhanced phosphorylation of the Na+-K+-2Cl- cotransporter NKCC1, suggesting that an increased activity of that transporter caused the early rise of intracellular Cl- levels. Immunohistochemistry of NKCC1 and KCC2, the main neuronal Cl- importer and exporter, respectively, exposed an inverse regulation by the inflammatory mediators. While the NKCC1 immunosignal increased, that of KCC2 declined after 3 hours of treatment. In contrast, the mRNA levels of the two transporters did not change markedly during this time. These data demonstrate a fundamental transition in Cl- homeostasis toward a state of augmented Cl- accumulation, which is induced by a 1–3 hour treatment with inflammatory mediators.ConclusionOur findings indicate that inflammatory mediators impact on Cl- homeostasis in DRG neurons. Inflammatory mediators raise intracellular Cl- levels and, hence, the driving force for depolarizing Cl- efflux. These findings corroborate current concepts for the role of Cl- regulation in the generation of inflammatory hyperalgesia and allodynia. As the intracellular Cl- concentration rises in DRG neurons, afferent signals can be boosted by excitatory Cl- currents in the presynaptic terminals. Moreover, excitatory Cl- currents in peripheral sensory endings may also contribute to the generation or modulation of afferent signals, especially in inflamed tissue.

Crystal structure of a light-driven sodium pump

Recently, the first known light-driven sodium pumps, from the microbial rhodopsin family, were discovered. We have solved the structure of one of them, Krokinobacter eikastus rhodopsin 2 (KR2), in the monomeric blue state and in two pentameric red states, at resolutions of 1.45 Å and 2.2 and 2.8 Å, respectively. The structures reveal the ion-translocation pathway and show that the sodium ion is bound outside the protein at the oligomerization interface, that the ion-release cavity is capped by a unique N-terminal α-helix and that the ion-uptake cavity is unexpectedly large and open to the surface. Obstruction of the cavity with the mutation G263F imparts KR2 with the ability to pump potassium. These results pave the way for the understanding and rational design of cation pumps with new specific properties valuable for optogenetics.

Differential maturation of chloride homeostasis in primary afferent neurons of the somatosensory system.

Recent research into the generation of hyperalgesia has revealed that both the excitability of peripheral nociceptors and the transmission of their afferent signals in the spinal cord are subject to modulation by Cl− currents. The underlying Cl− homeostasis of nociceptive neurons, in particular its postnatal maturation, is, however, poorly understood. Here we measure the intracellular Cl− concentration, [Cl−]i, of somatosensory neurons in intact dorsal root ganglia of mice. Using two‐photon fluorescence‐lifetime imaging microscopy, we determined [Cl−]i in newborn and adult animals. We found that the somatosensory neurons undergo a transition of Cl− homeostasis during the first three postnatal weeks that leads to a decline of [Cl−]i in most neurons. Immunohistochemistry showed that a major fraction of neurons in the dorsal root ganglia express the cation–chloride co‐transporters NKCC1 and KCC2, indicating that the molecular equipment for Cl− accumulation and extrusion is present. RT‐PCR analysis showed that the transcription pattern of electroneutral Cl− co‐transporters does not change during the maturation process.

Time-resolved photothermal methods: accessing time-resolved thermodynamics of photoinduced processes in chemistry and biology

Photothermal methods are currently being employed in a variety of research areas, ranging from materials science to environmental monitoring. Despite the common term which they are collected under, the implementations of these techniques are as diverse as the fields of application. In this review, we concentrate on the recent applications of time-resolved methods in photochemistry and photobiology.

The primary photoreaction of photoactive yellow protein (PYP): anisotropy changes and excitation wavelength dependence

Abstract The absorption and stimulated emission changes in the first 535 ps of the PYP photocycle can be described by four life times of 0.7, 6.3, and 220 ps and long lived. Two intermediates, I0 and I 0 ‡ , were identified. We did not obtain indications for a significant excitation wavelength dependent primary photochemistry as found in low temperature absorption spectroscopy. The anisotropy of the primary photoproduct I0 and its successor I 0 ‡ amounts to 0.3 – significantly lower than that of the bleached ground state (0.4). This distinctive change of the transition dipole moment orientation in the product state (24°) reflects changes of the chromophore geometry and electron density distribution caused by the photoisomerisation.

Photophysical study of a multi-chromophoric dendrimer by time-resolved fluorescence and femtosecond transient absorption spectroscopy

Abstract The photophysical properties of a dendrimer ( 1 ) bearing eight peryleneimide chromophores on a polyphenylene core were investigated by picosecond fluorescence and femtosecond transient absorption techniques. A peryleneimide attached to a hexaphenylbenzene unit ( 2 ) served as model compound. Multi-exponential fluorescence and fs-transient absorption decays were observed only for 1 , which could therefore be attributed to excimer-like interactions among neighbouring peryleneimides. The comparative time-resolved polarisation results on 1 and 2 indicate the occurrence of different depolarisation processes in the dendrimer assignable to the rotation of the molecule, a fractional motion maybe of a dendrimer branch and to an intramolecular energy transfer process.

Subunits act independently in a cyclic nucleotide‐activated K+ channel

Ion channels gated by cyclic nucleotides have crucial roles in neuronal excitability and signal transduction of sensory neurons. Here, we studied ligand binding of a cyclic nucleotide‐activated K+ channel from Mesorhizobium loti and its isolated cyclic nucleotide‐binding domain. The channel and the binding domain alone bind cyclic AMP with similar affinity in a non‐cooperative manner. The cAMP sensitivities of binding and activation coincide. Thus, each subunit in the tetrameric channel acts independently of the others. The binding and gating properties of the bacterial channel are distinctively different from those of eukaryotic cyclic nucleotide‐gated channels.