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As consequence of binding, gating of the channel is initiated. In the genome of the sea anemone Nematostella vectensis nv , a preferred model organism for the evolution of key bilaterian features, a TRPM2 ortholog has been identified that contains a NUDT9H domain as well.
An alternative ADPR binding site seems to be present but has not yet been characterized. All these manipulations create channels that are sensitive to hydrogen peroxide which fails to induce channel activity in wild-type nv TRPM2.
Thus, the two TRPM2 orthologs have evolved divergently but nevertheless gained analogous functional properties, i. The scientific community was very much excited when in , Perraud et al. Two characteristics of this channel, renamed in the meantime to TRPM2, were particularly fascinating.
The first one is its activation by ADPR, a metabolite which had not been on the list of potential stimuli of ion channels at this time, although it was known to induce the fertilization current in oocytes of the sea squirt Ciona intestinalis Wilding et al.
Few related substances have been reported to share its agonistic properties Grubisha et al. Experimentally, oxidative stress is frequently induced by extracellular application of hydrogen peroxide H 2 O 2 to the cells.
Consequently, current induction in response to H 2 O 2 takes some time, in contrast to the fast onset after stimulation of TRPM2 with high concentrations of intracellular ADPR during patch-clamp experiments. Until recently, it was believed that the catalytic activity of the respective enzyme domain contributes decisively to their gating process. The more C-terminally localized catalytic center is formed by a strongly conserved amino acid sequence, the so-called NUDIX-box Bessman et al.
It has been experimentally demonstrated that the two successive amino acid residues glutamate-phenylalanine of this region are especially important for the activity of the human enzyme Perraud et al. Taken together, there is ample and strong evidence for the notion that catalytic activity is not necessary and even detrimental for the activation of TRPM2. Alignment was performed using the tool at www. Several studies have clearly demonstrated that already subtle changes within the structure of the NUDT9H domain may lead to a complete loss of channel function e.
Obviously, binding and subsequent channel gating have very restricted structural requirements that can easily be disturbed. Methodological approaches that would provide a straightforward interpretation are not obvious. In this situation, a strategy may be helpful that has been successfully employed for several other ion channels: the structural and functional comparison of species variants. In this review, we will summarize the findings and perspectives gained from studies on the TRPM2 ortholog of the sea anemone Nematostella vectensis.
Of course, the question arises why choosing the sea anemone as species variant. There are several good reasons for this choice. First, the evolution especially of the TRPM channel subfamily seems to have taken a very interesting course. This and other indications allow the conclusion that in the beginning of the metazoan evolution, a TRPM2-like channel stepped on stage which probably represents the evolutionary ancestor of all modern TRPM channels.
Nematostella vectensis today represents a preferred model organism for the study of the evolution of some archetypal metazoan blueprints such as the immune system and the nervous system.
Especially for comparative studies on TRPM2, it is noteworthy that the natural habitats of Nematostella vectensis are salt marshes along the coasts of the northern Atlantic Ocean. Here, animals are commonly exposed to UV radiation and diverse chemicals, all of which can exert oxidative stress Tarrant et al. Because the mammalian TRPM2 ortholog represents a central player in the process of oxidative-stress mediated apoptosis, the suitability of Nematostella vectensis as a simplistic model appears evident.
However, and unexpectedly, this is achieved by vastly different mechanisms and parts of the channel protein. The genome of the starlet sea anemone Nematostella vectensis was sequenced and assembled by whole genome shotgun by Putnam et al.
As the complete expressed sequence tag start codon to stop codon open reading frame of the sea anemone TRPM2-like channel was published in the joint genome institute database jgi.
Notably, this region shows significant similarity to the corresponding region of the h TRPM3 channel which strengthens the hypothesis that a TRPM2-like channel represents a common ancestor of the contemporary TRPM-subfamily Mederos y Schnitzler et al. From the overall high topological similarity between nv TRPM2 and mammalian TRPs, we were confident in the beginning of our studies that the sea anemone ortholog could be functionally overexpressed with standard methods in mammalian cells, although until then only few examples existed where a successful heterologous expression of such far distantly related ion channels had been achieved and this was in oocytes of Xenopus laevis e.
The standard procedure of commercially available gene synthesis was used and the codon usage was adapted to the human expression system Ikemura, This manipulation is frequently a prerequisite for the successful heterologous expression of proteins from distantly related species.
This procedure was chosen to minimize the danger of artifacts due to species-specific antibodies. Wild-type and mutant nv TRPM2 channels were expressed in the plasma membrane with no obvious difference to the human ortholog. For functional analysis, mostly the variants without HA tags were studied using the standard whole-cell patch-clamp technique.
The non-electrophysiologists among the readers should understand that with this technique, the cytosol of the cells is replaced with the solution in the pipette within seconds by diffusion. Thus, the principal activators of h TRPM2 are effective in the ortholog of a distantly related species as well. These include in the first line the concentration-effect-relation as well as the on and off kinetics.
Both activation and inactivation kinetics are comparatively slow. At the end of the experiment, the currents were fully blocked by a substitution of extracellular monovalent cations with NMDG. C Similar experimental conditions as shown before but the cells had been pre-incubated for at least 5 min in bath solution containing 2-APB 0.
The rapid inactivation is completely suppressed. Single channel open times are extremely long in inside-out patches with ADPR on the cytosolic side of the plasma membrane, frequently reaching several hundreds of milliseconds. This may have not been expected anyway because they are extremely fast for a ligand-gated channel already at standard conditions.
Further evidence for this interpretation is discussed later in context of the effects of 2-APB. We therefore propose that the current decline should be referred to as inactivation because it relates to a pore-dependent mechanism.
The term desensitization should, in our opinion, not be used because it may be understood to describe a process that affects binding of ADPR for which no experimental indication exists.
Unfortunately, it is not easily possible to remove the stimulus ADPR during one experiment and repeat its application several times. In inside-out patches, this would be feasible; however, for some reasons that are not understood, single channel activity in response to ADPR persists much longer than whole-cell currents.
On the other hand, the compound is by no means channel-specific and its effect can be inhibitory as well as activating. This depends on its concentration and the channel type.
Even on one particular channel, it may exert both these opposite effects in a concentration dependent manner e. The potential of 2-APB in this respect has not yet been fully exploited but already the initial results reveal surprising insight, as well as they give rise to further questions and to hypotheses that should be tested in the near future. There are several key findings for 2-APB on nv TRPM2 that in combination result in a straightforward interpretation of its modes of action, although still some detailed questions remain open.
First, these are the peculiar on and off kinetics of 2-APB when used as a channel stimulus, i. There is a lag time of several tens of seconds before any effect can be observed but afterwards, the development of currents occurs very rapidly within seconds.
Thus, access to the pore is restricted as long as the channels are in a closed state but becomes fast as soon they are opened by 2-APB. As a result, an almost all-or-nothing kind of response to 2-APB is observed with an extremely steep concentration-response relation. While there is no apparent activation by 0.
The latter, however, takes place only after opening of the channel. A relatively small current is induced and inactivation takes place. Then, addition of 2-APB evokes currents with two remarkable properties. An extremely interesting process in nv TRPM2 is the fast current inactivation which discriminates it from its human ortholog. In nv TRPM2, inactivation takes place within fractions of a minute.
As molecular mechanisms for this phenomenon, the experiments with 2-APB and on the pore mutant QLP provide strong evidence that inactivation represents processes within the pore and should therefore, as noticed before, referred to as inactivation, rather than desensitization. Less clear is how this is prevented by 2-APB. In any case, the effects of 2-APB are immediately reversible after wash-out. As a side-note with the potential of an experimental pitfall, we would like to add that 2-APB may interfere with the large cation NMDG in a manner that is difficult to interpret biologically but may lead to incorrect conclusions in some experiments.
It is hoped that further comparison between nv TRPM2 and h TRPM2 and the study of pore chimeras will produce insight on the structural requirements that govern inactivation. A key feature of all TRPM2 channel orthologs studied previously which were all mammalian representatives without exception is their activation in response to oxidative stress Hara et al.
Currently the most accepted hypothesis is that H 2 O 2 activates the channel indirectly through an accumulation of intracellular ADPR Perraud et al. In extension of this view, a recent study reported that H 2 O 2 sensitizes h TRPM2 to the activation by physiological body temperatures; the sensitization is achieved by the oxidation of a methionine residue localized in the N-terminus of the channel Kashio et al.
This mechanism, under some experimental conditions and probably in vivo , may contribute to channel activation in response to oxidative challenges. The opposite findings were obtained. H 2 O 2 completely failed to induce any currents. There are quite a few point mutations that render channels completely insensitive to ADPR. Several short sequences were deleted or substituted with the same result Hara et al.
When analogous changes were introduced in nv TRPM2, again surprising findings were obtained. In no case, any change in the response to ADPR could be demonstrated.
However, these manipulations produced channels that were now readily activated by H 2 O 2. It is not worthwhile to summarize here the specific alterations of NUDT9H that were studied because it turned out that none of them contributes to the understanding of nv TRPM2 channel function. This latter role would fit very well to the two critical residues EF instead of IL in the enzymatic domain; furthermore, all changes that created H 2 O 2 sensitivity could then be interpreted as loss of ADPR degradation.
The absence of large parts of the C-terminus may lead to unpredictable structural changes of the protein, possibly resulting in misfolding and aberrant surface expression. Accordingly, it was mandatory to verify the correct surface expression of this variant. It is later discussed that incidentally, these expression studies revealed unforeseen insight into the function of the human NUDT9H domain. To begin with, the surface expression was almost normal which is prerequisite for further functional studies.
Oxidative stress leads to intracellular accumulation of ADPR. In contrast, nv TRPM2 lower, left exhibits full catalytic activity. Several questions immediately arise. This interpretation is in line with the experimental findings of Perraud et al. In principle, ADPR-dependent channel activation would not necessarily require a binding site. Alternatively, mechanisms like ADP-ribosylation should be discussed.
Moreover, ADPR is able to induce channel gating in cell-free patches. Potential binding sites might either exhibit known motifs for ADPR binding such as the Nudix box, or represent a new type of interaction. While Nudix box motifs cannot be found in the N-terminus or in other cytosolic parts of nv TRPM2, future search of binding sites may be guided by studies on a protein module ubiquitous in eukaryotes, bacteria, and archaea Chakravarthy et al.
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Интуиция подсказывала ему, что в глубинах дешифровального чудовища происходит что-то необычное. ГЛАВА 10 - Энсей Танкадо мертв? - Сьюзан почувствовала подступившую к горлу тошноту. - Вы его убили. Вы же сказали… - Мы к нему пальцем не притронулись, - успокоил ее Стратмор.
ГЛАВА 51 Джабба был похож на гигантского головастика. Подобно киноперсонажу, в честь которого он и получил свое прозвище, его тело представляло собой шар, лишенный всякой растительности. В качестве штатного ангела-хранителя компьютерных систем АН Б Джабба ходил по отделам, делал замечания, что-то налаживал и тем самым постоянно подтверждал свое кредо, гласившее, что профилактика-лучшее лекарство.
Ни один из поднадзорных ему компьютеров АНБ не заразился вирусом, и он был намерен не допустить этого и впредь.
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Раздался приглушенный звук выстрела. Мимо.
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Да, сэр. Уверен, что человеку вашего положения хорошо известно, что канадское правительство делает все для защиты соотечественников от неприятностей, которые случаются с ними в этих… э-э… скажем так, не самых передовых странах. Тонкие губы Клушара изогнулись в понимающей улыбке.