Differential function of the two nucleotide binding domains on cystic fibrosis transmembrane conductance regulator

AbstractThe genetic disease cystic fibrosis is caused by defects in the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR). CFTR belongs to the family of ABC transporters. In contrast to most other members of this family which transport substrates actively across a membrane, the main function of CFTR is to regulate passive flux of substrates across the plasma membrane. Chloride channel activity of CFTR is dependent on protein phosphorylation and presence of nucleoside triphosphates. From electrophysiological studies of CFTR detailed models of its regulation by phosphorylation and nucleotide interaction have evolved. These investigations provide ample evidence that ATP hydrolysis is crucial for CFTR gating. It becomes apparent that the two nucleotide binding domains on CFTR not only diverge strongly in sequence, but also in function. Based on previous models and taking into account new data from pre-steady-state experiments, a refined model for the action of nucleotides at two nucleotide binding domains was recently proposed

Dual Effects of Adp and Adenylylimidodiphosphate on Cftr Channel Kinetics Show Binding to Two Different Nucleotide Binding Sites

The CFTR chloride channel is regulated by phosphorylation by protein kinases, especially PKA, and by nucleotides interacting with the two nucleotide binding domains, NBD-A and NBD-B. Giant excised inside-out membrane patches from Xenopus oocytes expressing human epithelial cystic fibrosis transmembrane conductance regulator (CFTR) were tested for their chloride conductance in response to the application of PKA and nucleotides. Rapid changes in the concentration of ATP, its nonhydrolyzable analogue adenylylimidodiphosphate (AMP-PNP), its photolabile derivative ATP-P3-[1-(2-nitrophenyl)ethyl]ester, or ADP led to changes in chloride conductance with characteristic time constants, which reflected interaction of CFTR with these nucleotides. The conductance changes of strongly phosphorylated channels were slower than those of partially phosphorylated CFTR. AMP-PNP decelerated relaxations of conductance increase and decay, whereas ATP-P3-[1-(2-nitrophenyl)ethyl]ester only decelerated the conductance increase upon ATP addition. ADP decelerated the conductance increase upon ATP addition and accelerated the conductance decay upon ATP withdrawal. The results present the first direct evidence that AMP-PNP binds to two sites on the CFTR. The effects of ADP also suggest two different binding sites because of the two different modes of inhibition observed: it competes with ATP for binding (to NBD-A) on the closed channel, but it also binds to channels opened by ATP, which might either reflect binding to NBD-A (i.e., product inhibition in the hydrolysis cycle) or allosteric binding to NBD-B, which accelerates the hydrolysis cycle at NBD-A

Optogenetic Long-Term Manipulation of Behavior and Animal Development

Channelrhodopsin-2 (ChR2) is widely used for rapid photodepolarization of neurons, yet, as it requires high-intensity blue light for activation, it is not suited for long-term in vivo applications, e.g. for manipulations of behavior, or photoactivation of neurons during development. We used “slow” ChR2 variants with mutations in the C128 residue, that exhibit delayed off-kinetics and increased light sensitivity in Caenorhabditis elegans. Following a 1 s light pulse, we could photodepolarize neurons and muscles for minutes (and with repeated brief stimulation, up to days) with low-intensity light. Photoactivation of ChR2(C128S) in command interneurons elicited long-lasting alterations in locomotion. Finally, we could optically induce profound changes in animal development: Long-term photoactivation of ASJ neurons, which regulate larval growth, bypassed the constitutive entry into the “dauer” larval state in daf-11 mutants. These lack a guanylyl cyclase, which possibly renders ASJ neurons hyperpolarized. Furthermore, photostimulated ASJ neurons could acutely trigger dauer-exit. Thus, slow ChR2s can be employed to long-term photoactivate behavior and to trigger alternative animal development

Coupling the Cardiac Voltage-Gated Sodium Channel to Channelrhodopsin-2 Generates Novel Optical Switches for Action Potential Studies

Voltage-gated sodium (Na + ) channels respond to short membrane depolarization with conformational changes leading to pore opening, Na + influx, and action potential (AP) upstroke. In the present study, we coupled channelrhodopsin-2 (ChR2), the key ion channel in optogenetics, directly to the cardiac voltage-gated Na + channel (Na v 1.5). Fusion constructs were expressed in Xenopus laevis oocytes, and electrophysiological recordings were performed by the two-microelectrode technique. Heteromeric channels retained both typical Na v 1.5 kinetics and light-sensitive ChR2 properties. Switching to the current-clamp mode and applying short blue-light pulses resulted either in subthreshold depolarization or in a rapid change of membrane polarity typically seen in APs of excitable cells. To study the effect of individual K + channels on the AP shape, we co-expressed either K v 1.2 or hERG with one of the Na v 1.5-ChR2 fusions. As expected, both delayed rectifier K + channels shortened AP duration significantly. K v 1.2 currents remarkably accelerated initial repolarization, whereas hERG channel activity efficiently restored the resting membrane potential. Finally, we investigated the effect of the LQT3 deletion mutant ΔKPQ on the AP shape and noticed an extremely prolonged AP duration that was directly correlated to the size of the non-inactivating Na + current fraction. In conclusion, coupling of ChR2 to a voltage-gated Na + channel generates optical switches that are useful for studying the effect of individual ion channels on the AP shape. Moreover, our novel optogenetic approach provides the potential for an application in pharmacology and optogenetic tissue-engineering

Algen, Froscheier und Putzfimmel bei Fliegen

Zu den wichtigsten Signalmolekülen in lebenden Zellen gehört cAMP, das grundlegende Prozesse steuert. Wir haben kürzlich mithilfe eines Algenproteins eine neue Methode entwickelt, cAMP durch kurze Lichtblitze in tierischen Zellen und lebenden Tieren zu erzeugen. Bei Fliegen lassen sich damit Verhaltensänderungen an- und wieder abschalten. Die besonders hohe zeitliche Auflösung dieses neuen Lichtgesteuerten „Werkzeugs“ sollte es ermöglichen, die Rolle von cAMP bei komplexen biologischen Fragestellungen besser zu verstehen.Not Reviewe

Arthropod Diversity in Lama Forest Reserve (South Benin), a Mosaic of Natural, Degraded and Plantation Forests

Arthropod assemblages were examined in Lama forest reserve, a protected area situated in the Dahomey gap, southern Benin, composed of plantations, degraded forest and remnants of natural forest. The objectives were to compare assemblages in relation to forest type and use, to elucidate the value of forest plantations for biodiversity conservation and to identify indicator species for specific forest habitats. Arthropods were collected over an 11-month period, using standardized sets of traps (pitfall, emergence, Malaise and flight intercept traps). Nine different habitats were studied, including natural and degraded forest, forest plantations (Tectona grandis and Senna siamea) of different age, and isolated forest fragments. Our analysis focused on detritivorous and xylophagous arthropods but also included ground beetles and heteropterans, totalling 393 species. We found no differences in species richness among natural and degraded forest habitats in the centre of the reserve (Noyau central). Outside of the Noyau central, species richness was highest in old teak plantations and isolated forest fragments and lowest in young teak and fuelwood plantations. Detrended correspondence analysis (DCA) separated three main groups: (1) natural forest, (2) degraded forest and young plantations, and (3) old plantations and isolated forest fragments. Multiple regression of DCA scores of the first two axes on environmental variables identified one natural and three disturbance-related predictors of arthropod assemblages in Lama forest: soil type (texture), canopy height, naturalness (proportion of Guineo-Congolian plant species) and understorey vegetation cover. We identified 15 indicator species for six different forest habitats. The highest numbers were found in abandoned settlements and old teak plantations. β-diversity was similar among the three DCA ordination groups (degraded forest excluded). Values for β-diversity were relatively high, suggesting that all major forest habitats contribute significantly to regional species pools and should therefore be protected. To enhance arthropod diversity, we propose that management practices in Lama forest should aim to encourage the development of species-rich understorey vegetation of the Guineo-Congolian phytogeographical regio

Optogenetic tools for manipulation of cyclic nucleotides functionally coupled to cyclic nucleotide‐gated channels

Background and Purpose The cyclic nucleotides cAMP and cGMP are ubiquitous second messengers regulating numerous biological processes. Malfunctional cNMP signalling is linked to diseases and thus is an important target in pharmaceutical research. The existing optogenetic toolbox in Caenorhabditis elegans is restricted to soluble adenylyl cyclases, the membrane‐bound Blastocladiella emersonii CyclOp and hyperpolarizing rhodopsins; yet missing are membrane‐bound photoactivatable adenylyl cyclases and hyperpolarizers based on K+ currents. Experimental Approach For the characterization of photoactivatable nucleotidyl cyclases, we expressed the proteins alone or in combination with cyclic nucleotide‐gated channels in muscle cells and cholinergic motor neurons. To investigate the extent of optogenetic cNMP production and the ability of the systems to depolarize or hyperpolarize cells, we performed behavioural analyses, measured cNMP content in vitro, and compared in vivo expression levels. Key Results We implemented Catenaria CyclOp as a new tool for cGMP production, allowing fine‐control of cGMP levels. We established photoactivatable membrane‐bound adenylyl cyclases, based on mutated versions (“A‐2x”) of Blastocladiella and Catenaria (“Be,” “Ca”) CyclOp, as N‐terminal YFP fusions, enabling more efficient and specific cAMP signalling compared to soluble bPAC, despite lower overall cAMP production. For hyperpolarization of excitable cells by two‐component optogenetics, we introduced the cAMP‐gated K+‐channel SthK from Spirochaeta thermophila and combined it with bPAC, BeCyclOp(A‐2x), or YFP‐BeCyclOp(A‐2x). As an alternative, we implemented the B. emersonii cGMP‐gated K+‐channel BeCNG1 together with BeCyclOp. Conclusion and Implications We established a comprehensive suite of optogenetic tools for cNMP manipulation, applicable in many cell types, including sensory neurons, and for potent hyperpolarization.Deutsche Forschungsgemeinschaft (DFG) http://dx.doi.org/10.13039/501100001659Peer Reviewe