Kinetic properties and open probability of alpha7 nicotinic acetylcholine receptors.

The alpha7 nicotinic acetylcholine receptor (nAChR) has some peculiar kinetic properties. From the literature of alpha7 nAChR-mediated currents we concluded that experimentally measured kinetic properties reflected properties of the solution exchange system, rather than genuine kinetic properties of the receptors. We also concluded that all experimentally measured EC50 values for agonists must inherently be inaccurate. The aim of this study was to assess the undistorted kinetic properties of alpha7 nAChRs, and to construct an improved kinetic model, which can also serve as a basis of modeling the effect of the positive allosteric modulator PNU-120596, as it is described in the accompanying paper. Agonist-evoked currents were recorded from GH4C1 cells stably transfected with pCEP4/rat alpha7 nAChR using patch-clamp and fast solution exchange. We used two approaches to circumvent the problem of insufficient solution exchange rate: extrapolation and kinetic modeling. First, using different solution exchange rates we recorded evoked currents, and extrapolated their amplitude and kinetics to instantaneous solution exchange. Second, we constructed a kinetic model that reproduced concentration-dependence and solution exchange rate-dependence of receptors, and then we simulated receptor behavior at experimentally unattainably fast solution exchange. We also determined open probabilities during choline-evoked unmodulated and modulated currents using nonstationary fluctuation analysis. The peak open probability of 10 mM choline-evoked currents was 0.033 +/- 0.006, while in the presence of choline (10 mM) and PNU-120596 (10 muM), it was increased to 0.599 +/- 0.058. Our kinetic model could adequately reproduce low open probability, fast kinetics, fast recovery and solution exchange rate-dependent kinetics

Mode of action of the positive modulator PNU-120596 on alpha7 nicotinic acetylcholine receptors.

We investigated the mode of action of PNU-120596, a type II positive allosteric modulator of the rat alpha7 nicotinic acetylcholine receptor expressed by GH4C1 cells, using patch-clamp and fast solution exchange. We made two important observations: first, while PNU-120596 rapidly associated to desensitized receptors, it had at least hundredfold lower affinity to resting conformation, therefore at 10 muM concentration it dissociated from resting receptors; and second, binding of PNU-120596 slowed down dissociation of choline molecules from the receptor radically. We propose that when agonist concentration is transiently elevated in the continuous presence of the modulator (as upon the neuronal release of acetylcholine in a modulator-treated animal) these two elements together cause occurrence of a cycle of events: Binding of the modulator is limited in the absence of the agonist. When the agonist is released, it binds to the receptor, and induces desensitization, thereby enabling modulator binding. Modulator binding in turn traps the agonist within its binding site for a prolonged period of time. Once the agonist finally dissociated, the modulator can also dissociate without re-binding, and the receptor assumes its original resting conformation. In kinetic simulations this "trapped agonist cycle" mechanism did not require that the orthosteric and allosteric ligands symmetrically modify each other's affinity, only the modulator must decrease agonist accessibility, and the agonist must induce a conformation that is accessible to the modulator. This mechanism effectively prolongs and amplifies the effect of the agonist

Type I like behavior of the type II alpha 7 nicotinic acetylcholine receptor positive allosteric modulator A-867744

Cognitive impairment often involves the decreased expression or hypofunction of alpha 7-type nicotinic acetylcholine receptors (alpha 7 nAChRs). Agonists or positive allosteric modulators (PAMs) of alpha 7 nAChRs are known to be potential treatments for dementias, different neurodegenerative disorders, pain syndromes and conditions involving inflammation. In some of these conditions, it is desirable to maintain the temporal precision of fast cholinergic events, while in others, this temporal precision is unnecessary. For this reason, the optimal therapeutic effect for distinct indications may require PAMs with different mechanisms of action. The two major mechanisms are called "type I", which are compounds that augment alpha 7 nAChR-mediated currents but maintain their characteristic fast kinetics; and "type II", which are compounds that produce augmented and prolonged currents. In this study, we performed a kinetic analysis of two type II PAMs of the alpha 7 nAChR: PNU-120596 and A-867744, using a fast perfusion method that allowed high temporal resolution. We characterized the type of modulation produced by the two compounds, the state-dependence of the modulatory action, and the interaction between the two compounds. We found fundamental differences between the modulation mechanisms by PNU-120596 and A-867744. Most importantly, during brief agonist pulses, A-867744 caused a strikingly type I-like modulation, while PNU-120596 caused a type II-like prolonged activation. Our results demonstrate that specific compounds, even though all labeled as type II PAMs, can behave in completely different ways, including their onset and offset kinetics, state preference, and single channel open time. Our results emphasize that subtle details of the mechanism of action may be significant in assessing the therapeutic applicability of alpha 7 nAChR PAM compounds

The mechanism of non-blocking inhibition of sodium channels revealed by conformation-selective photolabeling

Sodium channel inhibitors can be used to treat hyperexcitability-related diseases, including epilepsies, pain syndromes, neuromuscular disorders, and cardiac arrhythmias. The applicability of these drugs is limited by their nonspecific effect on physiological function. They act mainly by sodium channel block and in addition by modulation of channel kinetics. While channel block inhibits healthy and pathological tissue equally, modulation can preferentially inhibit pathological activity. An ideal drug designed to target the sodium channels of pathological tissue would act predominantly by modulation. Thus far, no such drug has been described.Patch-clamp experiments with ultra-fast solution exchange and photolabeling-coupled electrophysiology were applied to describe the unique mechanism of riluzole on Nav1.4 sodium channels. In silico docking experiments were used to study the molecular details of binding.We present evidence that riluzole acts predominantly by non-blocking modulation. We propose that, being a relatively small molecule, riluzole is able to stay bound to the binding site, but nonetheless stay off the conduction pathway, by residing in one of the fenestrations. We demonstrate how this mechanism can be recognized.Our results identify riluzole as the prototype of this new class of sodium channel inhibitors. Drugs of this class are expected to selectively prevent hyperexcitability, while having minimal effect on cells firing at a normal rate from a normal resting potential

Az aktin citoszkeleton szabályozásának molekuláris aspektusai = Molecular Aspects of the Actin Cytoskeleton

Munkánk során számos - az aktin és az aktin citoszkeleton működésében fontos szerepet játszó - fehérje molekuláris tulajdonságait és kölcsönhatásait leírtuk. Folytattuk az aktin és miozin fehérjék kölcsönhatásának feltérképezését. Nemzetközi kooperációk keretei között gyors kinetikai módszereket alkalmazva jellemeztünk rovar (Drosophila) izomból származó miozint. Fluoreszcencia spektroszkópiai és kalorimetriai módszerekkel tanulmányoztuk az aktin kölcsönhatását toxikus peptidekkel (falloidin és jasplakinolid). A miozinok működésében meghatározó szerepet játszó nukleotidok, azokon belül is az ADP szerepét írtuk le részletesen egy összefoglaló cikkben. Vizsgáltuk a nem izom eredetű miozinok közül a patkányból származó miozin IX kinetikai tulajdonságait. Egy másik vizsgálatsorozatban a különböző emlős állatokból származó lassú és gyors miozin izoformák tulajdonságait írtuk le kinetikai módszerek segítségével. Ugyancsak nemzetközi együttműködés keretein belül tanulmányoztuk az aktinnak a forminokkal, a forminokon belül is az ún. Diaphanous Related forminokkal való kölcsönhatását. Megállapítottuk azt is, hogy az emlős (egér) sejtekből származó formin fragmentumok megváltoztatják az aktin filamentumok dinamikai tulajdonságait, konformációját. | We characterised a number of actin-binding proteins during the project. We described the dynamic aspects of the interaction between actin and myosin from rabbit skeletal muscle by using spectroscopic methods. Within the framework of international collaboration the properties of Drosophila flight muscle myosin was also investigated by rapid kinetic methods. We described the effect of toxic peptides (phalloidin and jasplakinolide) on the actin filaments by spectroscopic and calorimetric methods. By using rapid kinetic methods we studied the behaviour of a non-muscle myosin, rat myosin IX. In a separate study we described the differences between mysoin isoforms from various mammalian species. In another international collaboration we studied the interaction between actin and mammalian formins. We also showed by using specrtroscopic methods that these formin fragments change the dynamic and conformational properties of actin filaments

A nemszinaptikus nikotinikus acetilkolin és NMDA receptorok szerepe élettani körülmények között és pathológiás állapotokban = Role of nonsynaptic nicotinic acetylcholine receptors and NMDA receptors in physiological and pathophysiological conditions

A szélütés (stroke) utáni neurodegeneráció a jelenlegi morbiditási és mortalitási mutatók egyik legfontosabb tényezője. Az iszkémiás stroke kezelésében számos ígéretes gyógyszerjelölt molekula vallott kudarcot a klinikai vizsgálatokban. Ennek valószínűleg az az oka, hogy hiányosak ismereteink az iszkémiás kórképek kialakulásának mechanizmusaira vonatkozólag. A legtöbb központi idegrendszerre ható gyógyszert szinaptikusan elhelyezkedő receptorokra vagy transzporterekre fejlesztik annak érdekében, hogy igazán hatékony gyógyszereket tudjunk fejleszteni, figyelembe kell venni, hogy az extraszinaptikus receptorok és transzporterek száma jóval meghaladja a szinaptikusakét, illetve hogy nagyon sok központi idegrendszeri megbetegedés alapja a nemszinaptikus rendszer malfunkciója. Például, a szinaptikus NMDA receptorok aktivációja neuroprotektív hatást fejt ki, míg az extraszinaptikus NMDA receptor aktiváció excitotoxikus hatású. Konkrét javaslataink a gyógyszerfejlesztést illetően: Az NR2B alegységet tartalmazó NMDA receptorok szelektív gátlói (mint például a fluoxetine), és a nátriumcsatorna gátlók egyes típusai; mint neuroprotektív szerek. A nikotinikus agonisták pozitív modulátorai, amelyek a kognitív problémák kezelésében, ill. a dohányzásról való leszokás segítésében lehetnek hasznosak. | Neurodegeneration after a stroke is one of the major causes of present-day morbidity and mortality. There is a long list of neuroprotective compounds that have failed to be clinically useful in the treatment of ischaemic stroke. This is likely due, at least in part, to our inadequate knowledge regarding the core mechanisms of ischaemic diseases. Most “novel” drugs that target the CNS are designed to act on neurotransmitter receptors or transporters that are localised within synapses. To develop the most effective drugs, it is important to remember that there are extrasynaptic receptors and transporters that may outnumber those located within synapses and that, when malfunctioning, may be responsible for several symptoms of CNS disorders. For example, activation of synaptic NMDA receptors is neuroprotective, whereas stimulation of extrasynaptic NMDA receptors causes excitotoxicity. We suggest that future drug development research consider the following: Compounds that are able to selectively inhibit non-synaptic NR2B Glu receptors (such as Fluoxetine), and specific subtypes of sodium channel inhibitors as neuroprotective compounds. Positive modulators of nicotinic acetylcholine receptors. They would be potential drugs in the treatment of memory problems and in smoking cessation

Comparison of 2D and 3D neural induction methods for the generation of neural progenitor cells from human induced pluripotent stem cells

Neural progenitor cells (NPCs) from human induced pluripotent stem cells (hiPSCs) are frequently induced using 3D culture methodologies however, it is unknown whether spheroid-based (3D) neural induction is actually superior to monolayer (2D) neural induction. Our aim was to compare the efficiency of 2D induction with 3D induction method in their ability to generate NPCs, and subsequently neurons and astrocytes. Neural differentiation was analysed at the protein level qualitatively by immunocytochemistry and quantitatively by flow cytometry for NPC (SOX1, PAX6, NESTIN), neuronal (MAP2, TUBB3), cortical layer (TBR1, CUX1) and glial markers (SOX9, GFAP, AQP4). Electron microscopy demonstrated that both methods resulted in morphologically similar neural rosettes. However, quantification of NPCs derived from 3D neural induction exhibited an increase in the number of PAX6/NESTIN double positive cells and the derived neurons exhibited longer neurites. In contrast, 2D neural induction resulted in more SOX1 positive cells. While 2D monolayer induction resulted in slightly less mature neurons, at an early stage of differentiation, the patch clamp analysis failed to reveal any significant differences between the electrophysiological properties between the two induction methods. In conclusion, 3D neural induction increases the yield of PAX6(+)/NESTIN(+) cells and gives rise to neurons with longer neurites, which might be an advantage for the production of forebrain cortical neurons, highlighting the potential of 3D neural induction, independent of iPSCs' genetic background