Ion Channel gating

Ion Channel gating

We are searching data for your request:

Forums and discussions:
Manuals and reference books:
Data from registers:
Wait the end of the search in all databases.
Upon completion, a link will appear to access the found materials.

I have been studying ion channels and there is one thing i am confused about, gating between open and closed states in channels.

Am i right in thinking gating is so fast that it is effectively always open for ion flow? So that if ten ions flow through a channel, then one can imagine that the channel opens and closes so quickly it is basically always open for the flow of the ten ions, or does a channel open allow one ion through, close and then open again for the next ion?

There are several types of channels, but probably the type you are referring to is voltage gated ion channels.

The state in which these channels are, open or closed, depends on the action potential across the cell membrane.

If the potential is at the correct energy for the channel to be open it will remain open allowing a steady flow of ions through. Once enough ions have passed so that the potential across the membrane changes to meet the deactivation energy, the channel will close. It then enters a refractory period which requires repolarisation across the cell membrane so that the ion is reset ready to open and let ions through again.

Different ion channels have different activation potentials, deactivation potentials and different refractory periods. This results in different rates of opening and closing.

So based on the above, in answer to your question, no gating is not so fast that it is always open. There are significant periods of time where channels are closed allowing no flow of ions and significant periods of time where they are open allowing continuous flow of ions.

Deep-Channel uses deep neural networks to detect single-molecule events from patch-clamp data

Single-molecule research techniques such as patch-clamp electrophysiology deliver unique biological insight by capturing the movement of individual proteins in real time, unobscured by whole-cell ensemble averaging. The critical first step in analysis is event detection, so called “idealisation”, where noisy raw data are turned into discrete records of protein movement. To date there have been practical limitations in patch-clamp data idealisation high quality idealisation is typically laborious and becomes infeasible and subjective with complex biological data containing many distinct native single-ion channel proteins gating simultaneously. Here, we show a deep learning model based on convolutional neural networks and long short-term memory architecture can automatically idealise complex single molecule activity more accurately and faster than traditional methods. There are no parameters to set baseline, channel amplitude or numbers of channels for example. We believe this approach could revolutionise the unsupervised automatic detection of single-molecule transition events in the future.

Ion Channel gating - Biology

Ours is an interdisciplinary research program at the interface of chemistry and biology. We draw from disciplines as wide ranging as electrophysiology, lipidomics, protein chemistry and synthetic chemistry to address questions in mainly three areas summarized below.

A major thrust area of the group is to elucidate the molecular mechanisms underlying the opening and closing (gating) of ion channel proteins.

Ion channels are membrane proteins expressed in all cell types and are extremely important for life. For example, voltage-activated potassium and sodium channels expressed in neurons underlie the action potential which constitutes the nerve impulse. Other ion channels such as TRP channels are involved in a multitude of processes including thermal sensation and chemical sensing.

We have established the two-electrode voltage clamp (TEVC) and patch clamp electrophysiology set-ups in our lab and are employing them to study the gating of TRP and voltage-gated ion channels. We maintain a Xenopus laevis colony in our lab to procure oocytes for TEVC electrophysiology.

Another major focus of the laboratory is developing chemical biology-based approaches to study lipids.

There are thousands of lipids in cells, most with undefined function. One reason why lipids remain poorly understood as compared to proteins and nucleic acids is that powerful tools available to study them in cells are lacking.

We aim to address this urgent unmet requirement of efficacious approaches to study lipids by developing new technologies to label lipids in cells with synthetic, tailor‑made chemical handles, thereby endowing them with desired properties. We employ state-of-the art lipidomics (by using a triple quadrupole ion trap mass spectrometer that we have set up in our lab) and imaging approaches to rigorously characterize the resulting lipid labeling and employ these labeled lipids to discover lipid-interacting proteins by employing chemoproteomic approaches.

We are also actively working on developing efficacious methods for the site-specific labeling of biomolecules including proteins and lipids.

Projects on bioconjugation in the lab are organic chemistry-centric and involve the synthesis of organic compounds designed for orchestrating site-specific labeling of specific cellular biomolecules.

Mechanisms of pannexin1 channel gating and regulation

Pannexins are a family of integral membrane proteins with distinct post-translational modifications, sub-cellular localization and tissue distribution. Panx1 is the most studied and best-characterized isoform of this gene family. The ubiquitous expression, as well as its function as a major ATP release and nucleotide permeation channel, makes Panx1 a primary candidate for participating in the pathophysiology of CNS disorders. While many investigations revolve around Panx1 functions in health and disease, more recently, details started emerging about mechanisms that control Panx1 channel activity. These advancements in Panx1 biology have revealed that beyond its classical role as an unopposed plasma membrane channel, it participates in alternative pathways involving multiple intracellular compartments, protein complexes and a myriad of extracellular participants. Here, we review recent progress in our understanding of Panx1 at the center of these pathways, highlighting its modulation in a context specific manner. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.

Keywords: ATP Activation Channel Pannexin Panx1 Signaling.

Structural mechanisms for gating and ion selectivity of the human polyamine transporter ATP13A2

Mutations in ATP13A2, also known as PARK9, cause a rare monogenic form of juvenile onset Parkinson’s disease named Kufor-Rakeb syndrome and other neurodegenerative diseases. ATP13A2 encodes a neuroprotective P5B P-type ATPase highly enriched in the brain that mediates selective import of spermine ions from lysosomes into the cytosol via an unknown mechanism. Here we present three structures of human ATP13A2 bound to an ATP analogue or to spermine in the presence of phosphomimetics determined by electron cryo-microscopy. ATP13A2 autophosphorylation opens a lysosome luminal gate to reveal a narrow lumen access channel that holds a spermine ion in its entrance. ATP13A2’s architecture establishes physical principles underlying selective polyamine transport and anticipates a “pump-channel” intermediate that could function as a counter-cation conduit to facilitate lysosome acidification. Our findings establish a firm foundation to understand ATP13A2 mutations associated with disease and bring us closer to realizing ATP13A2’s potential in neuroprotective therapy.

Structures of the Parkinson’s disease-associated polyamine transporter ATP13A2

Structures of three transport cycle intermediates reveal the gating mechanism

Architecture of the polyamine binding site reveals mechanisms for ion selectivity

The polyamine binding site’s location anticipates an ion channel-like mechanism

Ion channel

Ion channels are pore-forming membrane proteins whose functions include establishing a resting membrane potential, shaping action potentials and other electrical signals by gating the flow of ions across the cell membrane, controlling the flow of ions across secretory and epithelial cells, .

ion channel
Any transmembrane protein complex that forms a water-filled channel across the phospholipid bilayer allowing selective ion transport down its electrochemical gradient. See also ion pump.
Full glossary .

Ion channels are present in the membranes that surround all biological cells. By conducting and controlling the flow of ions, these pore-forming proteins help establish the small negative voltage that all cells possess at rest (see cell potential).
1 Basic features .

Heart cell rhythm depends on the opening and closing of a complex series of valves on the cell membrane, called

s. Some valves let certain ions like potassium (K+) flow out, others let different ions like sodium (Na+) flow in. There are also pumps that actively move ions one direction or another.

s In The Daphnia Heart Biology Essay." 11 2013. All Answers Ltd. 04 2018 . Copy to Clipboard Reference Copied to Clipboard.

s have been extensively studied in excitatory cells like neurons and muscle fibers since the movement of ions across the membrane is an integral part of their function.

s /EYE-on/ n. Proteins, present in all cell membranes, governing the passage of specific ions between the interior and exterior of the cell.
ionize /EYE-ən-eyes/ (British: ionise) v. To change into ions.
IP₁ Inositol-1-phosphate.

that opens and closes to allow the cell to alter its membrane potential.
Gause's principle .

s are involved in a wide variety of biological processes and are a favorite target in the search for new drugs.
footer page .

in a membrane that opens and closes in response to changes in membrane potential (voltage) the sodium and potassium channels of neurons are examples.

s function as gated channels. These channels open or close depending on the presence or absence of a chemical or physical stimulus.
If chemical, the stimulus is a substance other than the one to be transported.

s which are permanently open
Retinal a photosensitive compound derived from beta-carotene.
Retinoate an oxidized derivative of retinol is formed by carboxylation of the aldehyde group of retinal.

A protein or assembly of several proteins in the cell membrane that opens and closes to let ions move in and out of cells. Ionising radiationRadiation that has so much energy that it can remove electrons from an atom's orbit when it comes into contact with it, 'ionising' or charging it.

s in neurons and other cells". Annu Rev Neurosci. 11: 119-36. doi:10.1146/ PMID 2452594.
^ Dulhunty A (2006). "Excitation-contraction coupling from the 1950s into the new millennium". Clin Exp Pharmacol Physiol. 33 (9): 763-72. doi:10.1111/j.1440-1681.

s open. At excitatory synapses, this opening allows positive ions to enter the neuron and results in depolarization of the membrane-a decrease in the difference in voltage between the inside and outside of the neuron.

Channel proteins that span the cell membrane form the

s. To determine the structure of proteins, scientists have often used X-ray crystallography.

These neurotransmitters diffuse across the very short gap from the axon to the surface of the target cell and bind to receptors that control

This magneto-thermal stimulation starts with mice that have been genetically engineered so the cells in the target neurons produce temperature-sensitive

s. Then, nanoparticles are injected into these parts of the brain, where they stick to the neurons.

Enzymes CD markers Blood group antigen proteins Nuclear receptors Transporters Ribosomal proteins G-protein coupled receptors Voltage-gated

s Predicted membrane proteins Predicted secreted proteins Predicted intracellular proteins Plasma proteins Transcription factors Mitochondrial proteins RNA polymerase .

Allows the passage of ions (Ca2+, Na+, Cl-) down their conc. gradient //passive - no ATP required
Some channels use a gate to regulate the flow of ions
Selective permeability - Not all molecules can pass through selective channels .

Types of receptors include

s that can open or close (gated) allowing ions to enter or leave cells, enzyme linked that activate endogenous or exogenous protein kinases (phosphate transferring enzymes), and serpentine or G-protein-linked.

The MCOLN1 gene codes for mucolipin1, which is a cat

that is non-selective. Pathological mutation involving this gene would therefore disrupt functions of this enzyme. This condition is inherited in an autosomal recessive pattern.
mucolipidin 1 deficiency .

Tiny fibers called tip links connect the top of each "hair" to its neighbor. The tip links are connected to stretch-sensitive

s you can think of the channels as tiny trap doors that when open let ions (charged molecules dissolved in the surrounding fluid) flow into the cell.

The receptors are transmitted-gated

s which open and let sodium and other positively charged ions into the postsynaptic neuron when the neurotransmitters bind. As these positively charged ions enter the postsynaptic neuron they cause its membrane to depolarize.

Arrival of the action potential causes some of the vesicles to move to the end of the axon and discharge their contents into the synaptic cleft. Released neurotransmitters diffuse across the cleft, and bind to receptors on the other cell's membrane, causing

REACTIVE OXYGEN SPECIES: Metabolism, Oxidative Stress, and Signal Transduction

Klaus Apel and Heribert Hirt
Vol. 55, 2004


▪ Abstract Several reactive oxygen species (ROS) are continuously produced in plants as byproducts of aerobic metabolism. Depending on the nature of the ROS species, some are highly toxic and rapidly detoxified by various cellular enzymatic and . Read More

Figure 1: Generation of different ROS by energy transfer or sequential univalent reduction of ground state triplet oxygen.

Figure 2: The principal features of photosynthetic electron transport under high light stress that lead to the production of ROS in chloroplasts and peroxisomes. Two electron sinks can be used to alle.

Figure 3: The principal modes of enzymatic ROS scavenging by superoxide dismutase (SOD), catalase (CAT), the ascorbate-glutathione cycle, and the glutathione peroxidase (GPX) cycle. SOD converts hydro.

Figure 4: Schematic depiction of cellular ROS sensing and signaling mechanisms. ROS sensors such as membrane-localized histidine kinases can sense extracellular and intracellular ROS. Intracellular RO.

Figure 5: Different roles of ROS under conditions of (a) pathogen attack or (b) abiotic stress. Upon pathogen attack, receptor-induced signaling activates plasma membrane or apoplast-localized oxidase.

New channel-gating mechanism discovered

When computational biophysicist Jianhan Chen at the University of Massachusetts Amherst and colleagues cracked the secret of how cells regulate Big Potassium (BK) channels, they thought it must be a computational artifact. But after many simulations and tests, they convinced themselves that they have identified the BK gating mechanism that had eluded science for many years.

Chen says, "The main way for the nervous system to send electrical signals is by opening and closing potassium and other ion channels that help regulate neuronal firing and neurotransmitter release. These Big Potassium channels are central for coupling electrical signaling to calcium-mediated events such as muscle contraction and neural excitation," and how blood pressure is regulated, for example.

"These BK channels contain extra-large pores, so they can sustain very large current, which lets the cell respond faster," he adds. BK channels play an important role in many health conditions such as hypertension, epilepsy, autism and mental retardation.

A key puzzle over the past 30 years has been trying to understand how cells close, or gate, BK channels, which have an unusually large central pore. In more typical-sized pores, the channel proteins generally contain structures that move into position to physically close the ion passage when ordered. But findings by others had shown that, inexplicably, the large central pore in BK channels "seems to remain wide open in both activated and deactivated states." Chen says, "The recently solved atomistic structures confirm that the pore still looks literally wide open even when it is closed to potassium passage. Nobody could understand this."

"There were a lot of hypotheses, but no answers," Chen notes. Now in Nature Communications, his team demonstrates that a physical gate is not required for closing BK channels. Instead, a phenomenon known as "hydrophobic dewetting" gives rise to a vapor phase in the pore's central cavity to block intracellular access to the selectivity filter.

Chen is part of UMass Amherst's chemistry, biochemistry and molecular biology program and a member of the campus's Institute for Applied Life Sciences, which translates fundamental science into new targets, leads and disease models. First author of their paper, Zhiguang Zhang, is a postdoctoral research fellow in Chen's lab and second author Mahdieh Yazdani is a graduate student there.

The gate mechanism in BK channels they have been studying is "drastically different from what has been observed in other ion channels," the authors point out. "We believe that this work represents a paradigm shift in our thinking of regulation and gating of BK channels," and is "one of the first few examples of a true 'hydrophobic gate,' where the barrier to ion permeation arises directly from dewetting transitions."

Hydrophobic dewetting refers to a phenomenon similar to the way water placed on an oily surface beads to form droplets. Initiation of dewetting transitions in BK channels requires changes in the pore shape and surface hydrophobicity driven by calcium binding. When the BK pore is oily, the water forms a vapor phase that acts like a barrier and prevents all ions from entering, Chen says. "Nothing gets through."

His team used computational modeling and physics-based atomistic simulations supported by the enormous computational power of a GPU cluster at the Massachusetts Green High Performance Computing Cluster in nearby Holyoke to carry out this work. They found the hydrophobic gating mechanism is also consistent with scanning mutagenesis studies showing that modulation of pore hydrophobicity is correlated with activation properties.

Chen explains, "We know the physical properties of each atom and how they interact. Our simulations put these systems together and from the collective dynamics we can examine how biological systems work." He adds, "Our data reconciles key results from previous experimental studies without invoking any crazy ideas. We are really proud of solving one of the biggest mysteries in the BK field."

He says, "If you think about why nature might want to use a vapor barrier where there is a big pore that has to carry a lot of electrical current, to apply a physical barrier you would need a protein structural re-arrangement which would probably be either too big or too slow, or both. In a way, nature is really clever in using this hydrophobic dewetting phenomenon to create a sensitive and fast gate. We were actually really surprised to see that the changes in pore shape and surface properties are relatively small and subtle, but they have big consequences on its hydration properties."

Further, Chen says, "In terms of understanding how the channel is gated, now we know more and it gives us a strong basis to see how other domains of BK channels talk to the pore and how the membrane voltage, calcium gradient, and a few other chemical signals control the state of the pore. In principle, that knowledge should be useful in developing new therapies and strategies in targeting the channel."

This work is the supported by a new four-year, $2.9 million grant recently funded by NIH's National Heart, Lung, and Blood Institute, to a collaborative team led by Jianmin Cui at Washington University, St. Louis, Chen at UMass Amherst and Xiaoqin Zou at the University of Missouri.

Adaptor proteins control ion channel gating mechanism

Scheme of the activation mechanism of TRPC4 and TRPC5 channels (left closed state, right open state). Channel proteins (blue), adaptor proteins (orange), PIP2 (green) and diacylglycerol (cyan) are involved in activation. Credit: M. Mederos, LMU

Ion channels are proteins that form pores in cellular membranes, which can be opened and shut like lock gates to allow the passage of electrically charged atoms (ions). Members of this class of proteins are crucial components involved in a wide range of processes that are essential for survival. In order to ensure that they correctly perform these functions, however, the opening and closing of these pores must be carefully regulated. LMU researchers led by Professor Dr. Michael Mederos y Schnitzler and Dr. Ursula Storch at the Walter Straub Institute for Pharmacology and Toxicology at LMU have now uncovered an activation mechanism in which an accessory molecular adaptor acts as a fail-safe mechanism to prevent inappropriate opening of two related ion channels. Their results have now been published in the journal PNAS.

The study focused on the ion channels TRPC4 und TRPC5, which belong to a particular family of cation channels. TRPC4 and 5 are found in a variety of cell types and tissues and, when activated, they selectively permit the passage of sodium (Na+) and calcium (Ca++) cations across cellular membranes. But how the two are activated has been unclear up to now. "The evidence available has been contradictory," says Mederos y Schnitzler. But he and his colleagues have now conclusively shown that TRPC4 and 5 are maintained in the inactive state by the binding of specific adaptor proteins, together with a small lipid molecule called PIP2. In both cases, enzymatic cleavage of PIP2 results in a specific change in the structural conformation of the channel, which in turn leads to dissociation of the adaptor. This process exposes a binding site on the TRPC protein for the intracellular messenger diacylglycerol (DAG), and attachment of DAG activates the channel, allowing cations to permeate though the pore.

The new findings clearly demonstrate that, contrary to what had been previously believed, both TRPC4 and TRPC5 are in fact sensitive to DAG. However, unlike all other members of the TRPC family of ion channels, TRPC4 and 5 cannot be activated directly by DAG, because their adaptor proteins dynamically regulate its access to the proteins, subsequent to the cleavage of PIP2. This is a novel role for an adaptor, as most serve as passive scaffolds for the assembly of protein complexes.

Depending on their subcellular localization, the adaptors that interact with TRPC4 and TRPC5 can promote or inhibit tumor cell growth, and since TRPC4 and 5 are expressed in many types of tumor cells, their interactions are also of medical relevance. This makes ion-channel/adaptor-protein complexes possible targets for anti-cancer drugs.

Ion Channel Gating

Ion channels are membrane proteins that allow transport of ions across otherwise impermeable membrane. Their dysfunction often results in severe hereditary diseases, underlying the importance of ion channels as pharmacological targets. Characterization of ion channel structure and function (gating) is a necessary step towards the design of their modulators. We are particularly interested in voltage-gated ion channels, which open or close an ion-selective pore via conformational changes that are triggered by changes in membrane voltage. We use molecular dynamics simulations, enhanced sampling methods and machine learning approaches corroborated by experiments from our collaborators ( Baron Chanda , Washington University – Peter Ruben , Simon Fraser University, Fredrik Elinder , Linköping University – Ann McDermott , Columbia University).

Voltage-gated sodium and potassium channels

Voltage-gated sodium and potassium channels are the main players in shaping the electrical signals called action potentials in neuronal and muscle cells. We study the voltage-dependent mechanism by which these channels transition between different states (resting, open and inactive) and we investigate the allosteric coupling between voltage-sensor domains (VSDs) and the central channel pore. We also study the channels’ modulation by perturbations from drugs and small molecules, lipids, mutations and post-translational modification.

KcsA potassium channel

KcsA is a pH-gated bacterial potassium channel. It was the first X-ray structure ever obtained of an ion channel. Due to its simplicity and high homology with potassium channels of higher organisms it is the ideal model system, the hydrogen atom of ion channels. Despite this apparent simplicity many aspects of its behavior are still highly debated. We study KcsA’s gating mechanisms, conductance and lipid-protein interactions.

Non domain-swapped ion channels

Non domain swapped voltage-gated ion channels gather different families of tetrameric channels which share a common specific architecture in which the voltage sensor and the pore domains of the same monomer are in contact with each other. Despite their similar 3D organisation, they display different mechanisms of gating. We are thus interested in understanding the coupling between the voltage sensor and pore domains within two main families – KCNH and HCN channels – to highlight common and different features by using and analyzing extensive/long MD simulations.

Perturbation by external electric fields

Voltage-gated ion channels normally operate under membrane voltages, which are on the order of 10-100 mV. However, when we expose cells to external electric fields, the membrane voltage can increase far beyond the physiological range and exceed several 100 mV. We can computationally predict voltage-sensing elements in any membrane protein, independent of its structure or function, by applying external electric fields in simulations. We are also interested in how high-intensity electric fields used in electroporation perturb the structure and function of different voltage-gated ion channels. This information is important for clinical applications where electroporation is used to transiently increase cell membrane permeability in order to enhance the intracellular delivery of therapeutic molecules.


Recent publications

Cannabidiol inhibits the skeletal muscle Nav1.4 by blocking its pore and by altering membrane elasticity MR Ghovanloo, K Choudhury, TS Bandaru, MA Fouda, K Rayani, R Rusinova, T Phaterpekar, K Nelkenbrecher, AR Watkins, D Poburko, J Thewalt, OS Andersen, L Delemotte, SJ Goodchild , PC Ruben.
J. Gen. Physiol., 2021 . DOI: 10.1085/jgp.202012701

Calmodulin acts as a state-dependent switch to control a cardiac potassium channel opening W Kang, AM Westerlund, J Shi,K McFarland White, AK Dou, AH Cui, JR Silva, Lucie Delemotte, J Cui.
Sci Adv., 2020 DOI: 10.1126/sciadv.abd6798

Helix breaking transition in the S4 of HCN channel is critical for hyperpolarization-dependent gating MA Kasimova, D Tewari, JB Cowgill, W Carrasquel Ursuleaz, JL Lin, L Delemotte, B Chanda.
eLife, 2019. DOI: 10.7554/eLife.53400

Determining the molecular basis of voltage sensitivity in membrane proteins MA Kasimova , E Lindahl , L Delemotte. J. Gen. Physiol, 2018. DOI: 10.1085/jgp.201812086

Gating interaction maps reveal a noncanonical electromechanical coupling mode in the Shaker K+ channel AI Fernández-Mariño, TJ Harpole, K Oelstrom, L Delemotte and B Chanda.
Nat. Struct. Mol. Biol., 2018. DOI: 10.1038/s41594-018-0047-3

Exploring the Viral Channel Kcv PBCV‑1 Function via Computation AEV Andersson, MA Kasimova, L Delemotte.
J. Memb. Biol., 2018. DOI: 10. 1007/s00 232-018-0022-2

Studying Kv Channels Function using Computational Methods A Deyawe, MA Kasimova, L Delemotte, G Loussouarn, M Tarek.
Potassium Channels, 2017. DOI: 10.1007/978-1-4939-7362-0_24

Does proton conduction in the voltage-gated H+ channel hHv1 involve grotthuss-like hopping via acidic residues? S C van Keulen, E Gianti, V Carnevale, ML Klein, U Rothlisberger and L Delemotte.
J. Phys. Chem. B, 2017 DOI: 10.1021/acs.jpcb.6b08339

Understanding TRPV1 activation by ligands: Insights from the binding modes of capsaicin and resiniferatoxin K Elokely, P Velisetty, L Delemotte, E Palovcak, ML Klein, T Rohacs, V Carnevale.
PNAS, 2016. DOI: 10.1073/pnas.1517288113

Gating pore currents are defects in common with two Nav1. 5 mutations in patients with mixed arrhythmias and dilated cardiomyopathy A Moreau, P Gosselin-Badaroudine, L Delemotte, ML Klein, M Chahine
The Journal of general physiology, 2015. DOI: 10.1085/jgp.201411304

Free-energy landscape of ion-channel voltage-sensor–domain activation L Delemotte, MA Kasimova, ML Klein, M Tarek, V Carnevale.
PNAS, 2015. DOI: 10.1073/pnas.1416959112


Molecular Biophysics Stockholm is a group of academic and industry researchers and engineers based at the Science for Life Laboratory in Solna, Stockholm County, Sweden.

Group members affiliated with KTH Royal Institute of Technology, Stockholm University, Karolinska Institute, and ERCO Pharma conduct computational and complementary structure-function studies on biomolecules.

Watch the video: Voltage-Gated Sodium Channels in Neurons (June 2022).


  1. Manolo

    I join. And I have faced it. Let's discuss this question. Here or in PM.

  2. Hebron

    all staff leave today?

  3. Cingeswiella


  4. Odi

    Yeah ... Life is like riding a bicycle. To keep your balance, you have to move.

  5. Vigor

    I consider, that you are mistaken. I suggest it to discuss.

Write a message