An all atomic, non-restrained molecular dynamics (MD) simulation in explicit water was used to study in detail the structural features of the highly conserved glycine-rich loop (GRL) of the α-subunit of the yeast mitochondrial processing peptidase (MPP) and its importance for the tertiary and quaternary conformation of MPP. Wild-type and GRL-deleted MPP structures were studied using non-restrained MD simulations, both in the presence and the absence of a substrate in the peptidase active site. Targeted MD simulations were employed to study the mechanism of substrate translocation from the GRL to the active site. We demonstrate that the natural conformational flexibility of the GRL is crucial for the substrate translocation process from outside the enzyme towards the MPP active site. We show that the α-helical conformation of the substrate is important not only during its initial interaction with MPP (i.e. substrate recognition), but also later, at least during the first third of the substrate translocation trajectory. Further, we show that the substrate remains in contact with the GRL during the whole first half of the translocation trajectory and that hydrophobic interactions play a major role. Finally, we conclude that the GRL acts as a precisely balanced structural element, holding the MPP subunits in a partially closed conformation regardless the presence or absence of a substrate in the active site.

• MeSH
• časové faktory MeSH
• glycin chemie   MeSH
• katalytická doména MeSH
• metaloendopeptidasy chemie   metabolismus   MeSH
• podjednotky proteinů chemie   metabolismus   MeSH
• Saccharomyces cerevisiae enzymologie   MeSH
• sekundární struktura proteinů MeSH
• simulace molekulární dynamiky MeSH
• substrátová specifita MeSH
• výpočetní biologie * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The transient receptor potential channel A1 (TRPA1) is unique among ion channels of higher vertebrates in that it harbors a large ankyrin repeat domain. The TRPA1 channel is expressed in the inner ear and in nociceptive neurons. It is involved in hearing as well as in the perception of pungent and irritant chemicals. The ankyrin repeat domain has special mechanical properties, which allows it to function as a soft spring that can be extended over a large range while maintaining structural integrity. A calcium-binding site has been experimentally identified within the ankyrin repeats. We built a model of the N-terminal 17 ankyrin repeat structure, including the calcium-binding EF-hand. In our simulations we find the calcium-bound state to be rigid as compared to the calcium-free state. While the end-to-end distance can change by almost 50% in the apo form, these fluctuations are strongly reduced by calcium binding. This increase in stiffness that constraints the end-to-end distance in the holo form is predicted to affect the force acting on the gate of the TRPA1 channel, thereby changing its open probability. Simulations of the transmembrane domain of TRPA1 show that residue N855, which has been associated with familial episodic pain syndrome, forms a strong link between the S4-S5 connecting helix and S1, thereby creating a direct force link between the N-terminus and the gate. The N855S mutation weakens this interaction, thereby reducing the communication between the N-terminus and the transmembrane part of TRPA1.

• MeSH
• ankyrinová repetice fyziologie   MeSH
• kationtové kanály TRP chemie   fyziologie   MeSH
• lidé MeSH
• molekulární modely MeSH
• motivy EF-ruky fyziologie   MeSH
• proteiny nervové tkáně chemie   fyziologie   MeSH
• simulace molekulární dynamiky MeSH
• vápník metabolismus   MeSH
• vápníkové kanály chemie   fyziologie   MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The ankyrin transient receptor potential channel TRPA1 is a non-selective cationic channel that is expressed by sensory neurons, where it can be activated by pungent chemicals, such as AITC (allyl isothiocyanate), cinnamon or allicin, by deep cooling (<18 °C) or highly depolarizing voltages (>+100 mV). From the cytoplasmic side, this channel can be regulated by negatively charged ligands such as phosphoinositides or inorganic polyphosphates, most likely through an interaction with as yet unidentified positively charged domain(s). In the present study, we mutated 27 basic residues along the C-terminal tail of TRPA1, trying to explore their role in AITC- and voltage-dependent gating. In the proximal part of the C-terminus, the function-affecting mutations were at Lys969, Arg975, Lys988 and Lys989. A second significant region was found in the predicted helix, centred around Lys1048 and Lys1052, in which single alanine mutations completely abolished AITC- and voltage-dependent activation. In the distal portion of the C-terminus, the charge neutralizations K1092A and R1099A reduced the AITC sensitivity, and, in the latter mutant, increased the voltage-induced steady-state responses. Taken together, our findings identify basic residues in the C-terminus that are strongly involved in TRPA1 voltage and chemical sensitivity, and some of them may represent possible interaction sites for negatively charged molecules that are generally considered to modulate TRPA1.

• MeSH
• aminokyseliny bazické genetika   fyziologie   MeSH
• ankyrinová repetice MeSH
• ankyriny MeSH
• ionty farmakologie   MeSH
• kationtové kanály TRP chemie   metabolismus   MeSH
• lidé MeSH
• membránové potenciály fyziologie   MeSH
• nervové receptory chemie   MeSH
• proteiny nervové tkáně chemie   metabolismus   MeSH
• statická elektřina MeSH
• substituce aminokyselin MeSH
• vápníkové kanály chemie   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The ankyrin transient receptor potential channel TRPA1 is a sensory neuron-specific channel that is gated by various proalgesic agents such as allyl isothiocyanate (AITC), deep cooling or highly depolarizing voltages. How these disparate stimuli converge on the channel protein to open/close its ion-conducting pore is unknown. We identify several residues within the S6 inner pore-forming region of human TRPA1 that contribute to AITC and voltage-dependent gating. Alanine substitution in the conserved mid-S6 proline (P949A) strongly affected the activation/deactivation and ion permeation. The P949A was functionally restored by substitution with a glycine but not by the introduction of a proline at positions -1, -2 or +1, which indicates that P949 is structurally required for the normal functioning of the TRPA1 channel. Mutation N954A generated a constitutively open phenotype, suggesting a role in stabilizing the closed conformation. Alanine substitutions in the distal GXXXG motif decreased the relative permeability of the channel for Ca(2+) and strongly affected its activation/deactivation properties, indicating that the distal G962 stabilizes the open conformation. G958, on the other hand, provides additional tuning leading to decreased channel activity. Together these findings provide functional support for the critical role of the putative inner pore region in controlling the conformational changes that determine the transitions between the open and close states of the TRPA1 channel.

• MeSH
• elektrofyziologie MeSH
• gating iontového kanálu fyziologie   MeSH
• isothiokyanatany farmakologie   MeSH
• kationtové kanály TRP metabolismus   MeSH
• lidé MeSH
• molekulární modely MeSH
• mutace genetika   MeSH
• potravinářské konzervační látky farmakologie   MeSH
• proteiny nervové tkáně metabolismus   MeSH
• substituce aminokyselin MeSH
• vápník metabolismus   MeSH
• vápníkové kanály metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH

Vaniloidy, aktivní složky některých pálivých rostlin, jakými jsou pálivá paprika a latex sukulentu Euphorbia resinifera, byly od nepaměti využívány k tišení bolestivých stavů. Přestože první písemné zmínky o využití vaniloidů se dochovaly již z počátku našeho letopočtu, dodnes přesně neznáme příčinu jejich analgetického účinku. Cílem tohoto příspěvku je informovat o současných poznatcích, které byly získaný o mechanizmech analgetického působení vaniloidů na buněčné a molekulární úrovni

Since time immemorial, people have used the effects of pungent vanilloid compounds such as resiniferatoxin and capsaicin for th e treatment of various painful disorders. Although the first written records on therapeutic use of vanilloids in pain treatment g o back thousands of years, the exact mechanisms of their analgesic action remain unclear. The aim of this summary is to provide a brie f overview of the current status of research on the antinociceptive effects of vanilloids at the cellular and molecular level.

• MeSH
• Capsicum fyziologie   metabolismus   účinky léků   MeSH
• finanční podpora výzkumu jako téma MeSH
• financování organizované MeSH
• fosforylace fyziologie   MeSH
• kapsaicin dějiny   terapeutické užití   MeSH
• kationtové kanály TRPV chemie   terapeutické užití   účinky léků   MeSH
• lidé MeSH
• měření bolesti psychologie   MeSH
• molekulární biologie metody   MeSH
• neopioidní analgetika farmakokinetika   farmakologie   terapeutické užití   MeSH
• práh bolesti fyziologie   psychologie   MeSH
• Check Tag
• lidé MeSH

• MeSH
• bolest farmakoterapie   MeSH
• desenzibilizace imunologická MeSH
• Euphorbia MeSH
• finanční podpora výzkumu jako téma MeSH
• financování organizované MeSH
• kafr terapeutické užití   MeSH
• kalmodulin MeSH
• kapsaicin farmakokinetika   terapeutické užití   MeSH
• kationtové kanály TRPV antagonisté a inhibitory   účinky léků   MeSH
• lidé MeSH
• Piper nigrum MeSH
• Check Tag
• lidé MeSH

Agonist-induced desensitization of the transient receptor potential vanilloid receptor-1 (TRPV1) is one of the key strategies that offer a way to alleviate neuropathic and inflammatory pain. This process is initiated by TRPV1 receptor activation and the subsequent entry of extracellular Ca(2+) through the channel into sensory neurones. One of the prominent mechanisms responsible for TRPV1 desensitization is dephosphorylation of the TRPV1 protein by the Ca(2+)/calmodulin-dependent enzyme, phosphatase 2B (calcineurin). Of several consensus phosphorylation sites identified so far, the most notable are two sites for Ca(2+)/calmodulin dependent kinase II (CaMKII) at which the dynamic equilibrium between the phosphorylated and dephosphorylated states presumably regulates agonist binding. We examined the mechanisms of acute Ca(2+)-dependent desensitization using whole-cell patch-clamp techniques in human embryonic kidney (HEK) 293T cells expressing the wild type or CaMKII phosphorylation site mutants of rat TRPV1. The nonphosphorylatable mutant S502A/T704I was capsaicin-insensitive but the S502A/T704A construct was fully functional, indicating a requirement for a specific residue at position 704. A point mutation at the nearby conserved residue R701 strongly affected the heat, capsaicin and pH-evoked currents. As this residue constitutes a stringent CaMKII consensus site but is also predicted to be involved in the interaction with membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)), these data suggest that in addition to dephosphorylation, or as its consequence, a short C-terminal juxtamembrane segment adjacent to the transient receptor potential box composed of R701 and T704 might be involved in the decelerated gating kinetics of the desensitized TRPV1 channel.

• MeSH
• elektrická stimulace metody   MeSH
• financování organizované MeSH
• fosfatidylinositol-4,5-difosfát farmakologie   MeSH
• kapsaicin farmakologie   MeSH
• kationtové kanály TRPV fyziologie   genetika   MeSH
• krysa rodu rattus MeSH
• lidé MeSH
• membránové potenciály MeSH
• metoda terčíkového zámku metody   MeSH
• mutace fyziologie   MeSH
• proteinkinasa závislá na vápníku a kalmodulinu typ 2 metabolismus   MeSH
• teplota MeSH
• transfekce MeSH
• transformované buněčné linie MeSH
• vápník MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• zvířata MeSH