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Ion channels affected by gravity

Ion channels affected by gravity


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In the literature I have found that action potentials behave differently when gravity is changed (cannot access fully).

Action potential properties are gravity dependent. http://link.springer.com/article/10.1007/BF02870977

Are there any ion channels in the neurons that are more or less likely to be sensitive to changes in gravity?


United in diversity: mechanosensitive ion channels in plants

Mechanosensitive (MS) ion channels are a common mechanism for perceiving and responding to mechanical force. This class of mechanoreceptors is capable of transducing membrane tension directly into ion flux. In plant systems, MS ion channels have been proposed to play a wide array of roles, from the perception of touch and gravity to the osmotic homeostasis of intracellular organelles. Three families of plant MS ion channels have been identified: the MscS-like (MSL), Mid1-complementing activity (MCA), and two-pore potassium (TPK) families. Channels from these families vary widely in structure and function, localize to multiple cellular compartments, and conduct chloride, calcium, and/or potassium ions. However, they are still likely to represent only a fraction of the MS ion channel diversity in plant systems.

Keywords: MCA MSL MscS TPK1 mechanotransduction.

Figures

Models for mechanosensitive (MS) ion…

Models for mechanosensitive (MS) ion channel gating. ( a ) The lipid-disordering model.…

Phylogenetic relationships, subcellular localizations, and…

Phylogenetic relationships, subcellular localizations, and topologies of MscS-like (MSL) channels. ( a )…

Molecularly uncharacterized mechanosensitive (MS) ion…

Molecularly uncharacterized mechanosensitive (MS) ion channel activities identified in plant membranes. Plasma membrane–localized…


Plants feel the force: How plants sense touch, gravity and other physical forces

"Picture yourself hiking through the woods or walking across a lawn," says Elizabeth Haswell, PhD, assistant professor of biology in Arts & Sciences at Washington University in St. Louis. "Now ask yourself: Do the bushes know that someone is brushing past them? Does the grass know that it is being crushed underfoot? Of course, plants don't think thoughts, but they do respond to being touched in a number of ways."

"It's clear," Haswell says, "that plants can respond to physical stimuli, such as gravity or touch. Roots grow down, a 'sensitive plant' folds its leaves, and a vine twines around a trellis. But we're just beginning to find out how they do it," she says.

In the 1980s, work with bacterial cells showed that they have mechanosensitive channels, tiny pores in the cells membrane that open when the cell bloats with water and the membrane is stretched, letting charged atoms and other molecules rush out of the cell. Water follows the ions, the cell contracts, the membrane relaxes, and the pores close.

Genes encoding seven such channels have been found in the bacterium Escherichia coli and 10 in Arabidopsis thaliana, a small flowering plant related to mustard and cabbage. Both E. coli and Arabidopsis serve as model organisms in Haswell's lab.

She suspects that there are many more channels yet to be discovered and that they will prove to have a wide variety of functions.

Recently, Haswell and colleagues at the California Institute of Technology, who are co-principal investigators on an National Institutes of Health (NIH) grant to analyze mechanosensitive channels, wrote a review article about the work so far in order to "get their thoughts together" as they prepared to write the grant renewal. The review appeared in the Oct. 11 issue of Structure.

Swelling bacteria might seem unrelated to folding leaflets, but Haswell is willing to bet they're all related and that mechanosensitive ion channels are at the bottom of them all. After all, plant movements -- both fast and slow -- are ultimately all hydraulically powered where ions go the water will follow.

Giant E. coli cells

The big problem with studying ion channels has always been their small size, which poses formidable technical challenges.

Early work in the field, done to understand the ion channels whose coordinated opening and closing creates a nerve impulse, was done in exceptionally large cells: the giant nerve cells of the European squid, which had projections big enough to be seen with the unaided eye.

Experiments with these channels eventually led to the development of a sensitive electrical recording technique known as the patch clamp that allowed researchers to examine the properties of a single ion channel. Patch clamp recording uses as an electrode a glass micropipette that has an open tip. The tip is small enough that it encloses a "patch" of cell membrane that often contains just one or a few ion channels.

Patch clamp work showed that there were many different types of ion channels and that they were involved not just in the transmission of nerve impulses but also with many other biological processes that involve rapid changes in cells.

Mechanosensitive channels were discovered when scientists started looking for ion channels in bacteria, which wasn't until the 1980s because ion channels were associated with nerves and bacteria weren't thought to have a nervous system.

In E. coli, the ion channels are embedded in the plasma membrane, which is inside a cell wall, but even if the wall could be stripped away, the cells are far too small to be individually patched. So the work is done with specially prepared giant bacterial cells called spherophlasts.

These are made by culturing E. coli in a broth containing an antibiotic that prevents daughter cells from separating completely when a cell divides. As the cells multiply, "snakes" of many cells that share a single plasma membrane form in the culture. "If you then digest away the cell wall, they swell up to form a large sphere," Haswell says.

Not that spheroplasts are that big. "We're doing most of our studies in Xenopus oocytes (frog eggs), whose diameters are 150 times bigger than those of spheroplasts," she says.

Three mechanosensitive channel activities

To find ion channels in bacteria, scientists did electrophysiological surveys of spheroplasts. They stuck a pipette onto the spheroplast and applied suction to the membrane as they looked for tiny currents flowing across the membrane.

"What they found was really amazing," Haswell says. "There were three different activities that are gated (triggered to open) only by deformation of the membrane." (They were called "activities" because nobody knew their molecular or genetic basis yet.)

The three activities were named mechanosensitive channels of large (MscL), small (MscS) and mini (MscM) conductance. They were distinguished from one another by how much tension you had to introduce in order to get them to open and by their conductance.

One of the labs working with spheroplasts was led by Ching Kung, PhD, at the University of Wisconsin-Madison. The MscL protein was identified and its gene was cloned in 1994 by Sergei Sukharev, PhD, then a member of Kung's lab. His tour-de-force experiment, Haswell says, involved reconstituting fractions of the bacterial plasma membrane into synthetic membranes (liposomes) to see whether they would confer large-channel conductance.

In 1999, the gene encoding MscS was identified in the lab of Ian Booth, PhD, at the University of Aberdeen. Comparatively, little work has been done on the mini channel, which is finicky and often doesn't show up, Haswell says, though a protein contributing to MscM activity was recently identified by Booth's group.

Once both genes were known, researchers did knockout experiments to see what happened to bacteria that didn't have the genes needed to make the channels. What they found, says Haswell, was that if both the MscL and MscS genes were missing, the cells could not survive "osmotic downshock," the bacterial equivalent of water torture.

"The standard assay," Haswell says, "is to grow the bacteria for a couple of generations in a very salty broth, so that they have a chance to balance their internal osmolyte concentration with the external one." (Osmolytes are molecules that affect osmosis, or the movement of water into and out of the cell.) "They do this," she says, "by taking up osmolytes from the environment and by making their own."

"Then," she says, "you take these bacteria that are chockfull of osmolytes and throw them into fresh water. If they don't have the MscS and MscL proteins that allow them to dump ions to avoid the uncontrolled influx of water, they don't survive." It's a bit like dumping saltwater fish into a freshwater aquarium.

Why are there three mechanosenstivie channel activities? The currently accepted model, Haswell says is that the channels with the smaller conductances are the first line of defense. They open early in response to osmotic shock so that the channel of large conductance, through which molecules the cell needs can escape, doesn't open unless it is absolutely necessary. The graduated response thus gives the cell its best chance for survival.

Crystallizing the proteins

The next step in this scientific odyssey, figuring out the proteins' structures, also was very difficult. Protein structures are traditionally discovered by purifying a protein, crystallizing it out of a water solution, and then bombarding the crystal with X-rays. The positions of the atoms in the protein can be deduced from the X-ray diffraction pattern.

In a sense crystallizing a protein isn't all that different from growing rock candy from a sugar solution, but, as always, the devil is in the details. Protein crystals are much harder to grow than sugar crystals and, once grown, they are extremely fragile. They even can even be damaged by the X-ray probes used to examine them.

And to make things worse MscL and MscS span the plasma membrane, which means that their ends, which are exposed to the periplasm outside the cell and the cytoplasm inside the cell, are water-loving and their middle sections, which are stuck in the greasy membrane, are repelled by water. Because of this double nature it is impossible to precipitate membrane proteins from water solutions.

Instead the technique is to surround the protein with what have been characterized as "highly contrived detergents," that protect them -- but just barely -- from the water. Finding the magical balance can take as long as a scientific career.

The first mechanosensitive channel to be crystallized was MscL -- not the protein in E. coli but the analogous molecule (a homolog) from the bacterium that causes tuberculosis. This work was done in the lab of one of Haswell's co-authors, Douglas C. Rees a Howard Hughes investigator at the California Institute of Technology.

MscS from E. coli was crystallized in the Rees laboratory several years later, in 2002, and an MscS protein with a mutation that left it stuck in the presumed open state was crystallized in the Booth laboratory in 2008. "So now we have two crystal structures for MscS and two (from different bacterial strains) for MscL," Haswell says.

Of plants and mutants

Up to this point, mechanosensitive channels might not seem all that interesting because the lives of bacteria are not of supreme interest to us unless they are making us ill.

However, says, Haswell, in the early 2000s, scientists began to compare the genes for the bacterial channels to the genomes of other organisms and they discovered that there are homologous sequences not just in other bacteria but also in some multicellular organisms, including plants.

"This is where I got involved," she says. "I was interested in gravity and touch response in plants. I saw these papers and thought these homologs were great candidates for proteins that might mediate those responses."

"There are 10 MscS-homologs in Arabidopsis and no MscL homologs," she says. "What's more, different homologs are found not just in the cell membrane but also in chloroplast and mitochondrial membranes. "

The chloroplast is the light-capturing organelle in a plant cell and the mitochondria is its power station both are thought to be once-independent organisms that were engulfed and enslaved by cells which found them useful. Their membranes are vestiges of their free-living past.

The number of homologs and their locations in plant cells suggests these channels do much more than prevent the cells from taking on board too much water.

So what exactly were they doing? To find out Haswell got online and ordered Arabidopsis seeds from the Salk collection in La Jolla, Calif., each of which had a mutation in one of the 10 channel genes.

From these mutants she's learned that two of the ten channels control chloroplast size and proper division as well as leaf shape. Plants with mutations in these two MscS channel homologs have giant chloroplasts that haven't divided properly. The monster chloroplasts garnered her lab the cover of the August issue of The Plant Cell.

"We showed that bacteria lacking MscS and MscL don't divide properly either,"Haswell says, "so the link between these channels and division is evolutionarily conserved."

The big idea

But Haswell and her co-authors think they are only scratching the surface. "We are basing our understanding of this class of channels on MscS itself, which is a very reduced form of the channel," she says. "It's relatively tiny."

"But we know that some of the members of this family have long extensions that stick out from the membrane either outside or inside the cell. We suspect this means that the channels not only discharge ions, but that they also signal to the whole cell in other ways. They may be integrated into common signaling pathways, such as the cellular osmotic stress response pathway.

We think we may be missing a lot of complexity by focusing too exclusively on the first members of this family of proteins to be found and characterized," she says. "We think there's a common channel core that makes these proteins respond to membrane tension but that all kinds of functionally relevant regulation may be layered on top of that."

"For example," she says, "there's a channel in E. coli that's closely related to MscS that has a huge extension outside the cell that makes it sensitive to potassium. So it's a mechanosensitive channel but it only gates in the presence of potassium. What that's important for, we don't yet know, but it tells us there are other functions out there we haven't studied."

What about the sensitive plant?

So are these channels at the bottom of the really fast plant movements like the sensitive plant's famous touch shyness? (To see a movie of this and other "nastic" (fast) movements, go to the Plants in Motion site maintained by Haswell's colleague Roger P. Hangartner of Indiana University).

Haswell is circumspect. "It's possible," she says. "In the case of Mimosa pudica there's probably an electrical impulse that triggers a loss of water and turgor in cells at the base of each leaflet, so these channel proteins are great candidates.


Life on Earth is used to gravity – so what happens to our cells and tissues in space?

Look ma, no gravity! Credit: NASA , CC BY

There's one force whose effects are so deeply entrenched in our everyday lives that we probably don't think much about it at all: gravity. Gravity is the force that causes attraction between masses. It's why when you drop a pen, it falls to the ground. But because gravitational force is proportional to the mass of the object, only large objects like planets create tangible attractions. This is why the study of gravity traditionally focused on massive objects like planets.

Our first manned space missions, however, completely changed how we thought about gravity's effects on biological systems. The force of gravity doesn't just keep us anchored to the ground it influences how our bodies work on the smallest of scales. Now with the prospect of longer space missions, researchers are working to figure out what a lack of gravity means for our physiology – and how to make up for it.

Freed from gravity's grip

It wasn't until explorers traveled to space that any earthly creature had spent time in a microgravity environment.

Scientists observed that returning astronauts had grown taller and had substantially reduced bone and muscle mass. Intrigued, researchers started comparing blood and tissue samples from animals and astronauts before and after space travel to assess the impact of gravity on physiology. Astronaut-scientists in the largely gravity-free environment of the International Space Station began to investigate how cells grow while in space.

Most experiments in this field are actually conducted on Earth, though, using simulated microgravity. By spinning objects – such as cells – in a centrifuge at fast speeds, you can create these reduced gravity conditions.

Our cells have evolved to deal with forces in a world characterized by gravity if they're suddenly liberated from gravity's effects, things start getting strange.

On months-long expeditions in space, astronauts’ bodies have to deal with a gravity-free environment very different to what they’re used to on Earth. Credit: NASA, CC BY

Detecting forces at a cellular level

Along with the force of gravity, our cells are also subjected to additional forces, including tension and shear stresses, as conditions change within our bodies.

Our cells need ways to sense these forces. One of the widely accepted mechanisms is through what are called mechano-sensitive ion channels. These channels are pores on the cell membrane that let particular charged molecules pass in or out of the cell depending on the forces they detect.

An example of this kind of mechano-receptor is the PIEZO ion channel, found in almost all cells. They coordinate touch and pain sensation, depending on their locations in the body. For instance, a pinch on the arm would activate a PIEZO ion channel in a sensory neuron, telling it to open the gates. In microseconds, ions such as calcium would enter the cell, passing on the information that the arm got pinched. The series of events culminates in withdrawal of the arm. This kind of force-sensing can be crucial, so cells can quickly react to environmental conditions.

Without gravity, the forces acting on mechano-sensitive ion channels are imbalanced, causing abnormal movements of ions. Ions regulate many cellular activities if they're not going where they should when they should, the work of the cells goes haywire. Protein synthesis and cellular metabolism are disrupted.

Physiology without gravity

Over the past three decades, researchers have carefully teased out how particular kinds of cells and body systems are affected by microgravity.

Channels in a cell’s membrane act as gatekeepers, opening or closing to let molecules in or out in response to a particular stimulus. Credit: Efazzari, CC BY-SA
  • Brain: Since the 1980s, scientists have observed that the absence of gravity leads to enhanced blood retention in the upper body, and so increased pressure in the brain. Recent research suggests this heightened pressure reduces the release of neurotransmitters, key molecules that brain cells use to communicate. This finding has motivated studies into common cognitive problems, such as learning difficulties, in returning astronauts.
  • Bone and muscle: The weightlessness of space can cause more than a 1 percent bone loss per month, even in astronauts who undergo stringent exercise regimes. Now scientists are using advances in genomics (the study of DNA sequences) and proteomics (the study of proteins) to identify how bone cells' metabolism is regulated by gravity. In the absence of gravity, scientists have found that the type of cells in charge of bone formation are suppressed. At the same time the type of cells responsible for degrading bone are activated. Together it adds up to accelerated bone loss. Researchers have also identified some of the key molecules that control these processes.
  • Immunity: Spacecraft are subject to rigorous sterilization to prevent transfer of foreign organisms. Nevertheless, during the Apollo 13 mission, an opportunistic pathogen infected astronaut Fred Haise. This bacteria, Pseudomonas aeruginosa, usually infects only immune-compromised individuals. This episode triggered more curiosity about how the immune system adapts to space. By comparing astronauts' blood samples before and after their space missions, researchers discovered that the lack of gravity weakens the functions of T-cells. These specialized immune cells are responsible for fighting a range of diseases, from the common cold to deadly sepsis.

Compensating for the lack of gravity

NASA and other space agencies are investing to support strategies that will prepare humans for longer-distance space travel. Figuring out how to withstand microgravity is a big part of that.

The current best method to overcome the absence of gravity is to increase load on the cells in another way – via exercise. Astronauts typically spend at least two hours each day running and weight-lifting to maintain healthy blood volume and reduce bone and muscle loss. Unfortunately, rigorous exercises can only slow down the deterioration of the astronauts' health, not prevent it completely.

Supplements are another method researchers are investigating. Through large-scale genomics and proteomics studies, scientists have managed to identify specific cell-chemical interactions affected by gravity. We now know that gravity affects key molecules that control cellular processes like growth, division and migration. For instance, neurons grown in microgravity on the International Space Station have fewer of one kind of receptor for the neurotransmitter GABA, which controls motor movements and vision. Adding more GABA restored function, but the exact mechanism is still unclear.

NASA is also evaluating whether adding probiotics to space food to boost the digestive and immune systems of astronauts may help stave off the negative effects of microgravity.

In early days of space travel, one of the first challenges was figuring out how to overcome gravity so a rocket could break free of Earth's pull. Now the challenge is how to offset the physiological effects of a lack of gravitational force, especially during long space flights.

This article was originally published on The Conversation. Read the original article.


Physiology without gravity

Brain: Since the 1980s, scientists have observed that the absence of gravity leads to enhanced blood retention in the upper body, and so increased pressure in the brain. Recent research suggests this heightened pressure reduces the release of neurotransmitters, key molecules that brain cells use to communicate. This finding has motivated studies into common cognitive problems, such as learning difficulties, in returning astronauts.

Bone and muscle: The weightlessness of space can cause more than a 1 percent bone loss per month, even in astronauts who undergo stringent exercise regimes. Now scientists are using advances in genomics (the study of DNA sequences) and proteomics (the study of proteins) to identify how bone cells’ metabolism is regulated by gravity. In the absence of gravity, scientists have found that the type of cells in charge of bone formation are suppressed. At the same time the type of cells responsible for degrading bone are activated. Together it adds up to accelerated bone loss. Researchers have also identified some of the key molecules that control these processes.


Genetic Defects of Ion Channels

Several genetic diseases exhibiting defects in the physiological functions of ion channels have now been shown to be caused by mutations in the genes coding for specific ion channels. For example, a cardiac potassium channel named HERG (human ether-a-go-go-related gene) acts to protect the heart against inappropriate rhythmicity. People lacking a functional HERG gene exhibit an abnormality on their electrocardiogram called "long Q-T syndrome," which predisposes them to sudden cardiac arrest when they are under stress. Cystic fibrosis results from mutations of a particular chloride channel called the cystic fibrosis transmembrane conductance regulator.


How plants sense touch, gravity and other physical forces

Elizabeth Haswell, Ph.D., assistant professor of biology in Arts & Sciences at Washington University in St. Louis, in a growth chamber with her "lab rats," Arabidopsis plants she uses to understand how plants respond to touch, gravity and other mechanical forces. If wild-type Arabidopsis plants are touched frequently. their growth is stunted. Credit: David Kilper/WUSTL

(PhysOrg.com) -- At the bottom of plants' ability to sense touch, gravity or a nearby trellis are mechanosensitive channels, pores through the cells' plasma membrane that are opened and closed by the deformation of the membrane. Elisabeth Haswell, Ph.D., a biologist at Washington University in St. Louis, is studying the roles these channels play in Arabdopsis plants by growing mutant plants that lack one or more of the 10 possible channel proteins in this species.

"Picture yourself hiking through the woods or walking across a lawn," says Elizabeth Haswell, PhD, assistant professor of biology in Arts & Sciences at Washington University in St. Louis. "Now ask yourself: Do the bushes know that someone is brushing past them? Does the grass know that it is being crushed underfoot? Of course, plants don't think thoughts, but they do respond to being touched in a number of ways."

"It's clear," Haswell says, "that plants can respond to physical stimuli, such as gravity or touch. Roots grow down, a 'sensitive plant' folds its leaves, and a vine twines around a trellis. But we're just beginning to find out how they do it," she says.

In the 1980s, work with bacterial cells showed that they have mechanosensitive channels, tiny pores in the cells membrane that open when the cell bloats with water and the membrane is stretched, letting charged atoms and other molecules to rush out of the cell. Water follows the ions, the cell contracts, the membrane relaxes, and the pores close.

Genes encoding seven such channels have been found in the bacterium Escherichia coli and 10 in Arabidopsis thaliana, a small flowering plant related to mustard and cabbage. Both E. coli and Arabidopsis serve as model organisms in Haswell's lab.

She suspects that there are many more channels yet to be discovered and that they will prove to have a wide variety of functions.

Recently, Haswell and colleagues at the California Institute of Technology, who are co-principal investigators on an National Institutes of Health (NIH) grant to analyze mechanosensitive channels, wrote a review article about the work so far in order to "get their thoughts together" as they prepared to write the grant renewal. The review appeared in the Oct. 11 issue of Structure.

Swelling bacteria might seem unrelated to folding leaflets, but Haswell is willing to bet they're all related and that mechanosensitive ion channels are at the bottom of them all. After all, plant movements — both fast and slow — are ultimately all hydraulically powered where ions go the water will follow.

Giant E. coli cells

The big problem with studying ion channels has always been their small size, which poses formidable technical challenges.

Early work in the field, done to understand the ion channels whose coordinated opening and closing creates a nerve impulse, was done in exceptionally large cells: the giant nerve cells of the European squid, which had projections big enough to be seen with the unaided eye.

Experiments with these channels eventually led to the development of a sensitive electrical recording technique known as the patch clamp that allowed researchers to examine the properties of a single ion channel. Patch clamp recording uses as an electrode a glass micropipette that has an open tip. The tip is small enough that it encloses a "patch" of cell membrane that often contains just one or a few ion channels.

Patch clamp work showed that there were many different types of ion channels and that they were involved not just in the transmission of nerve impulses but also with many other biological processes that involve rapid changes in cells.

Mechanosensitive channels were discovered when scientists started looking for ion channels in bacteria, which wasn't until the 1980s because ion channels were associated with nerves and bacteria weren't thought to have a nervous system.

In E. coli, the ion channels are embedded in the plasma membrane, which is inside a cell wall, but even if the wall could be stripped away, the cells are far too small to be individually patched. So the work is done with specially prepared giant bacterial cells called spherophlasts.

These are made by culturing E. coli in a broth containing an antibiotic that prevents daughter cells from separating completely when a cell divides. As the cells multiply, "snakes" of many cells that share a single plasma membrane form in the culture. "If you then digest away the cell wall, they swell up to form a large sphere," Haswell says.

Not that spheroplasts are that big. "We're doing most of our studies in Xenopus oocytes (frog eggs), whose diameters are 150 times bigger than those of spheroplasts," she says.

Three mechanosensitive channel activites

To find ion channels in bacteria, scientists did electrophysiological surveys of spheroplasts. They stuck a pipette onto the spheroplast and applied suction to the membrane as they looked for tiny currents flowing across the membrane.

"What they found was really amazing," Haswell says. "There were three different activities that are gated (triggered to open) only by deformation of the membrane." (They were called "activities" because nobody knew their molecular or genetic basis yet.)

The three activities were named mechanosensitive channels of large (MscL), small (MscS) and mini (MscM) conductance. They were distinguished from one another by how much tension you had to introduce in order to get them to open and by their conductance.

One of the labs working with spheroplasts was led by Ching Kung, PhD, at the University of Wisconsin-Madison. The MscL protein was identified and its gene was cloned in 1994 by Sergei Sukharev, PhD, then a member of Kung's lab. His tour-de-force experiment, Haswell says, involved reconstituting fractions of the bacterial plasma membrane into synthetic membranes (liposomes) to see whether they would confer large-channel conductance.

Representations of the pore section of the MscS channel in E. coli in its nonconducting (top) and open (bottom) configurations are based on X-ray crystallization studies of the protein’s structure. The transition between closed and open states is often described as similar to the narrowing and expanding of the pupil of the eye. The “closed” state can still appear to have an opening because amino acids around the opening act as a “hydrophobic plug” that prevents ions from moving through it.

In 1999, the gene encoding MscS was identified in the lab of Ian Booth, PhD, at the University of Aberdeen. Comparatively, little work has been done on the mini channel, which is finicky and often doesn't show up, Haswell says, though a protein contributing to MscM activity was recently identified by Booth's group.

Once both genes were known, researchers did knockout experiments to see what happened to bacteria that didn't have the genes needed to make the channels. What they found, says Haswell, was that if both the MscL and MscS genes were missing, the cells could not survive "osmotic downshock," the bacterial equivalent of water torture.

"The standard assay," Haswell says, "is to grow the bacteria for a couple of generations in a very salty broth, so that they have a chance to balance their internal osmolyte concentration with the external one." (Osmolytes are molecules that affect osmosis, or the movement of water into and out of the cell.) "They do this," she says, "by taking up osmolytes from the environment and by making their own."

"Then," she says, "you take these bacteria that are chockfull of osmolytes and throw them into fresh water. If they don't have the MscS and MscL proteins that allow them to dump ions to avoid the uncontrolled influx of water, they don't survive." It's a bit like dumping saltwater fish into a freshwater aquarium.

Why are there three mechanosenstivie channel activities? The currently accepted model, Haswell says is that the channels with the smaller conductances are the first line of defense. They open early in response to osmotic shock so that the channel of large conductance, through which molecules the cell needs can escape, doesn't open unless it is absolutely necessary. The graduated response thus gives the cell its best chance for survival.

E. coli’s MscL and MscS channels (left), says Haswell, may be reduced forms of mechanosensitive channels. Many of the other known channels have extensions either outside or inside the cell that suggest they are up to something more complex than MscL and MscS. (The small brown clothespins between blue squiggles are a schematic representation of the plasma membrane the channels bridge.)

Crystallizing the proteins

The next step in this scientific odyssey, figuring out the proteins' structures, also was very difficult. Protein structures are traditionally discovered by purifying a protein, crystallizing it out of a water solution, and then bombarding the crystal with X-rays. The positions of the atoms in the protein can be deduced from the X-ray diffraction pattern.

In a sense crystallizing a protein isn't all that different from growing rock candy from a sugar solution, but, as always, the devil is in the details. Protein crystals are much harder to grow than sugar crystals and, once grown, they are extremely fragile. They even can even be damaged by the X-ray probes used to examine them.

And to make things worse MscL and MscS span the plasma membrane, which means that their ends, which are exposed to the periplasm outside the cell and the cytoplasm inside the cell, are water-loving and their middle sections, which are stuck in the greasy membrane, are repelled by water. Because of this double nature it is impossible to precipitate membrane proteins from water solutions.

Instead the technique is to surround the protein with what have been characterized as "highly contrived detergents," that protect them — but just barely — from the water. Finding the magical balance can take as long as a scientific career.

The first mechanosensitive channel to be crystallized was MscL—not the protein in E. coli but the analogous molecule (a homolog) from the bacterium that causes tuberculosis. This work was done in the lab of one of Haswell's co-authors, Douglas C. Rees a Howard Hughes investigator at the California Institute of Technology.

MscS from E. coli was crystallized in the Rees laboratory several years later, in 2002, and an MscS protein with a mutation that left it stuck in the presumed open state was crystallized in the Booth laboratory in 2008. "So now we have two crystal structures for MscS and two (from different bacterial strains) for MscL," Haswell says.

Of plants and mutants

Up to this point, mechanosensitive channels might not seem all that interesting because the lives of bacteria are not of supreme interest to us unless they are making us ill.

However, says, Haswell, in the early 2000s, scientists began to compare the genes for the bacterial channels to the genomes of other organisms and they discovered that there are homologous sequences not just in other bacteria but also in some multicellular organisms, including plants.

"This is where I got involved," she says. "I was interested in gravity and touch response in plants. I saw these papers and thought these homologs were great candidates for proteins that might mediate those responses."

"There are 10 MscS-homologs in Arabidopsis and no MscL homologs," she says. "What's more, different homologs are found not just in the cell membrane but also in chloroplast and mitochondrial membranes. "

The chloroplast is the light-capturing organelle in a plant cell and the mitochondria is its power station both are thought to be once-independent organisms that were engulfed and enslaved by cells which found them useful. Their membranes are vestiges of their free-living past.

The number of homologs and their locations in plant cells suggests these channels do much more than prevent the cells from taking on board too much water.

So what exactly were they doing? To find out Haswell got online and ordered Arabidopsis seeds from the Salk collection in La Jolla, Calif., each of which had a mutation in one of the 10 channel genes.

From these mutants she's learned that two of the ten channels control chloroplast size and proper division as well as leaf shape. Plants with mutations in these two MscS channel homologs have giant chloroplasts that haven't divided properly. The monster chloroplasts garnered her lab the cover of the August issue of The Plant Cell.

"We showed that bacteria lacking MscS and MscL don't divide properly either,"Haswell says, "so the link between these channels and division is evolutionarily conserved."

But Haswell and her co-authors think they are only scratching the surface. "We are basing our understanding of this class of channels on MscS itself, which is a very reduced form of the channel," she says. "It's relatively tiny."

"But we know that some of the members of this family have long extensions that stick out from the membrane either outside or inside the cell. We suspect this means that the channels not only discharge ions, but that they also signal to the whole cell in other ways. They may be integrated into common signaling pathways, such as the cellular osmotic stress response pathway.

We think we may be missing a lot of complexity by focusing too exclusively on the first members of this family of proteins to be found and characterized," she says. "We think there's a common channel core that makes these proteins respond to membrane tension but that all kinds of functionally relevant regulation may be layered on top of that."

"For example," she says, "there's a channel in E. coli that's closely related to MscS that has a huge extension outside the cell that makes it sensitive to potassium. So it's a mechanosensitive channel but it only gates in the presence of potassium. What that's important for, we don't yet know, but it tells us there are other functions out there we haven't studied."

What about the sensitive plant?

So are these channels at the bottom of the really fast plant movements like the sensitive plant's famous touch shyness? (To see a movie of this and other "nastic" (fast) movements, go to the Plants in Motion site maintained by Haswell's colleague Roger P. Hangartner of Indiana University).

Haswell is circumspect. "It's possible," she says. "In the case of Mimosa pudica there's probably an electrical impulse that triggers a loss of water and turgor in cells at the base of each leaflet, so these channel proteins are great candidates.


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Mechanosensitive channels are activated by stress in the actin stress fibres, and could be involved in gravity sensing in plants

H. Tatsumi, Department of Physiology, Nagoya University Graduate School of Medicine, 65 Tsurumai Showa-ku, Nagoya Aichi 4668550, Japan.

EcoTopia Science Institute, Nagoya University, Nagoya, Japan

Department of Biology, Tokyo Gakugei University, Tokyo, Japan

Graduate School of Biological Science, Nara Institute of Science and Technology, Nara, Japan

FIRST Research Center for Innovative Nanobiodevice, Nagoya University, Nagoya, Japan

Nagoya University Graduate School of Medicine, Nagoya, Japan

Department of Biology, Tokyo Gakugei University, Tokyo, Japan

Nagoya University Graduate School of Medicine, Nagoya, Japan

H. Tatsumi, Department of Physiology, Nagoya University Graduate School of Medicine, 65 Tsurumai Showa-ku, Nagoya Aichi 4668550, Japan.

EcoTopia Science Institute, Nagoya University, Nagoya, Japan

Department of Biology, Tokyo Gakugei University, Tokyo, Japan

Graduate School of Biological Science, Nara Institute of Science and Technology, Nara, Japan

FIRST Research Center for Innovative Nanobiodevice, Nagoya University, Nagoya, Japan

Nagoya University Graduate School of Medicine, Nagoya, Japan

Department of Biology, Tokyo Gakugei University, Tokyo, Japan

Abstract

Mechanosensitive (MS) channels are expressed in a variety of cells. The molecular and biophysical mechanism involved in the regulation of MS channel activities is a central interest in basic biology. MS channels are thought to play crucial roles in gravity sensing in plant cells. To date, two mechanisms have been proposed for MS channel activation. One is that tension development in the lipid bilayer directly activates MS channels. The second mechanism proposes that the cytoskeleton is involved in the channel activation, because MS channel activities are modulated by pharmacological treatments that affect the cytoskeleton. We tested whether tension in the cytoskeleton activates MS channels. Mammalian endothelial cells were microinjected with phalloidin-conjugated beads, which bound to stress fibres, and a traction force to the actin cytoskeleton was applied by dragging the beads with optical tweezers. MS channels were activated when the force was applied, demonstrating that a sub-pN force to the actin filaments activates a single MS channel. Plants may use a similar molecular mechanism in gravity sensing, since the cytoplasmic Ca 2+ concentration increase induced by changes in the gravity vector was attenuated by potential MS channel inhibitors, and by actin-disrupting drugs. These results support the idea that the tension increase in actin filaments by gravity-dependent sedimentation of amyloplasts activates MS Ca 2+ -permeable channels, which can be the molecular mechanism of a Ca 2+ concentration increase through gravistimulation. We review recent progress in the study of tension sensing by actin filaments and MS channels using advanced biophysical methods, and discuss their possible roles in gravisensing.


The Relative Refractory Period:

Immediately after the absolute refractory period, the cell can generate an action potential, but only if it is depolarized to a value more positive than normal threshold. This is true because some sodium channels are still inactive and some potassium channels are still open. This is called the relative refractory period. The cell has to be depolarized to a more positive membrane potential than normal threshold to open enough sodium channels to begin the positive feedback loop. The lengths of the absolute and relative refractory periods are important because they determine how fast neurons can generate action potentials.

The neuron is a cell with electric activity. It is based on the idea that neuronal activity can be completely described by the flow of different currents associated with the neuron's membrane. The membrane of the cell has an electric potential Vm called membrane potential and is assumed equal at all points of the membrane. The presence of such an electric potential at the membrane of the neuron is the result of the charges balancing between the internal and external environment of the cell. Several types of ions of either positive or negative charge are present outside and inside the cell, and the difference between inner and the outer concentration of the different ion species produces the polarization of the membrane. The membrane potential is measured in Volts (V). The electric activity of a neuron is due to the continuous exchanges of electric currents or charges with other neurons.

To understand the generation of sodium and potassium currents with respect to the action potential generation with the help of remotely triggered equipment.

Hardware neuron model can provide real time processing. By going through the circuit dynamics one can understand both biological as well as physiological behaviors of neuron.

We have designed an analog neuron model using Resistors, transistors, capacitors and externally input voltage. These all are some basic electronic components which will make analog neuron to behave like normal neuron.

  • Resistance represents the difficulty a particle experiences while moving in a medium. It is measured in ohms. The inverse of resistance is conductance. Conductance is the ease at which a particle can move through a medium. It is measured in Siemens. Because they are inversely related, high conductance are correlated to low resistance, and vice versa. It is important to note that generally speaking resistance and conduction in the neuron are dealing with the ability of ions to cross the membrane. Thus it often referred to as membrane resistance or membrane conductance. As such, when the majority of ion channels are closed, few ions cross the membrane, and membrane resistance is said to be high.
  • The capacitor is a passive electronic components consisting of pair of conductors separated by an insulator. The cell membrane is also said to act as a capacitor, and has a property known as capacitance. A capacitor consists of two conducting regions separated by an insulator. A capacitor works by accumulating a charge on one of the conducting surfaces. As this charge builds, it creates an electric field that pushes like charges on the other side of the insulator away. This causes an induced current known as a capacitive current. It is important to realize that there is no current between the conducting surfaces of the capacitor. Capacitance may be defined two ways as:

Thus given a set number of charges on each side of the membrane, a higher capacitance results in a lower potential difference. In a cellular sense, increased capacitance requires a greater ion concentration difference across the membrane.

  • Transistor is an active semiconductor device commonly used to amplify (strengthen) or switch electronic signal. Here we are using 3 transistors, two NPN and one PNP transistor. Transistor has mainly three terminals. Emitter (E), Base (B) and Collector(C). Transistor T1 and T3 are NPN transistor and T2 is a PNP transistor. For an NPN transistor collector voltage is more positive than emitter. So current flows from collector to emitter. For a PNP transistor emitter voltage is more positive than collector. So current flows from emitter to collector.

We have added one diode at the base of T1 to eliminate the bias voltage of T2. Strictly speaking it limits the fast inward current to a short burst.

Here we give an input excitation to the cell membrane as square wave form of amplitude 2Volt peak to peak (Vpp), since we want to obtain the output as pulse wave form. A square wave resembles to an impulse wave form in shape when pulse width is low. Here R1 represent a variable resistor which represent the membrane resistance and is inversely proportional to membrane conductance. By varying this R1 membrane conductance can be changed considerably i.e., when membrane resistance (R1) decreases the membrane conductance increases making flow of signals easier. Cm is the membrane capacitance. In any cell membrane there is a charge separation across the cell. The seperation of charge by a insulator causes a capacitive effect on the cell. This effect is modelled as membrane capacitance. If there is only the resistor when the input voltage is applied, then voltage will be changed to steady state value, hence we are using a capacitor Cm along with it which resist this change. When the applied input makes the membrane capacitance to change above threshold value, then only voltage gated sodium channels open. The membrane potential is measured with respect to ground.


When the input excitation is given the membrane capacitance Cm begins to charge, when the voltage across the capacitor reaches more than cut in voltage of transistor T1, the transistor turns on and the current flows from collector to emitter. Then the base voltage of transistor T2 becomes less and T2 also turns on and current flows from emitter to collector i.e., Na channel is on and INa begins to flow inwardly. The energy for it provided by an electrical gradient of Na + across the membrane, here it is modelled as ENa.


The threshold value of potassium channels is modeled as transistor base emitter cut in voltage. The sodium current charges the capacitor C1. When the voltage across the capacitor C1 reaches more than cut in voltage of transistor T3, the transistor turns on and the potassium current flows from collector to emitter outwardly i.e., K channels on. Thus the depolarising phase of an action potential.

By this time membrane capacitor Cm becomes fully charged and begins to discharge i.e., when the capacitor voltage drops transistor T1 turn off, consequently T2. Then sodium current stops its flow i.e., sodium channel closes. As a result capacitor C1 begins to discharge and transistor T3 turn which leads potassium current to stop flow. Thus the repolarising phase of an action potential.

To study the effects of drugs like TEA or TTX on the ion channels.

Some chemical agents can selectively block voltage-dependent membrane channels. Tetrodoxin (TTX), which comes from the Japanese puffer fish, blocks the voltage-dependent changes in Na + permeability, but has no effect on the voltage-dependent changes in K + permeability. This observation indicates that the Na + and K + channels are unique one of these can be selectively blocked and not affect the other. Another agent, tetraethylammonium (TEA), has no effect on the voltage-dependent changes in Na+ permeability, but it completely abolishes the voltage-dependent changes in K + permeability.


Watch the video: Ion channels (September 2022).


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