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Shown here are a normal round worm (top) and a
constipated round worm (bottom). The arrow points to the
constipated worm's gut, which is swollen with waste because
scientists crippled the worm's ability to use protons like
neurotransmitters.
Image by
Paola Nix, University of Utah.
The top diagram shows the cross-section of worm,
including muscles that surround the intestine to help the worm
defecate. The bottom diagram shows how protons act like
neurotransmitters. The protons are pumped out of the intestine and
then bind to receptors on the surrounding muscle.
Image by Glen Ernstrom, University of Utah.
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Not only did the researchers show protons can
act like neurotransmitters, they identified the genes and proteins
involved in the process in round worms, which are about 1 millimeter
(a 25th of an inch) long and also are known as nematodes.
Previous research indicated the brains of humans and mice also have
proton pumps and receptors to move protons between cells. The new
study raises the possibility those protons may be transmitting nerve
signals in the brain, says Jorgensen and study co-author Wayne Davis,
a research assistant professor of biology.
“This is the first time we have found protons acting as transmitters,”
Davis says. “It could be that these processes occur in humans. There
are proton pumps present in intestinal cells and in the brain of
humans and mice. Some of the pumps are thought to make acid for the
gut to digest food. But why are proton pumps in the brain"”
Jorgensen adds: “Mice lacking the proton receptor cannot learn. It may
be that the proton pump and receptor are required for learning,” and
thus protons may act like neurotransmitters in the brain.
Utah graduate student Asim Beg (now at Columbia University) conducted
the study with Jorgensen, Davis, graduate student Paola Nix and
postdoctoral researcher Glen Ernstrom.
A Discovery from Constipated Worms
Atoms are made of nuclei that contain positively charged protons and
uncharged neutrons, orbited by negatively charged electrons. So
protons are among the smallest components of matter.
Previously recognized neurotransmitters such as serotonin (known for
its role in preventing depression) and dopamine (involved in addiction
to cocaine and other drugs) are more than 100 times larger than
protons. That makes protons “the world’s smallest transmitter,” says
Jorgensen, also a Howard Hughes Medical Institute investigator.
Acids burn because they contain high concentrations of protons, which
already were known to help the stomach digest food.
The new study shows that at least in some circumstances, protons also
may be used by cells to communicate. “Normally you think of proton
concentration increasing to digest food,” Davis says. “Now we see the
cell is using these protons to communicate. The protons are acting
like a word in a language that cells use to talk to each other.”
When the study began, nobody thought a new transmitter would be
identified. Instead, the researchers were trying to understand how
worms poop. Why" “Eating, moving, having sex and pooping are common
things that all animals do,” says Davis.
Nematodes, or Caenorhabditis elegans, have about 1,000 cells and are
simple animals studied by researchers worldwide. Nematodes have many
of the same tissues – nerves, muscle and intestine – that are found in
humans, and most of the same genes, making the worm a model for
studying human biology.
Defecation in round worms is surprisingly complex. The animal has
muscle contractions every 50 seconds that result in expulsion of
intestinal contents. The 50-second cycle is an example of a biological
clock, like those that regulate wake-sleep cycles and many other
behaviors in animals.
“We were interested in teasing apart the components required for [defecation]
clock function,” says Nix. “To do that we searched for mutations that
affected the clock.”
By exposing worms to chemicals that altered their DNA, the researchers
found mutant worms that couldn’t defecate or had trouble doing so.
“The worms are constantly eating, so if they don’t poop regularly,
they become very constipated,” says Nix.
In the round worm’s tail, muscles surround the tube-shaped intestine,
and there is a fluid-filled space between the intestine and the
surrounding muscles. The muscles contract to help the worm defecate.
The researchers identified two different gene mutations that prevented
such contractions.
Proteins and Protons Propel Pooping Process
One gene, named pbo-4 (for posterior body contraction), produces a
protein that pumps protons out of the intestine, acting “like a
revolving door where sodium is allowed in [the gut] while protons go
out [of the gut] into the fluid-filled space that is very close to the
surrounding muscle,” says Davis.
The second gene, pbo-5, makes a receptor protein on the muscles that
surround the gut. After the protons are released from the intestine,
they bind to the receptor protein.
“The receptor acts like an ear that allows the muscle to hear that
protons are present,” says Beg. The receptor opens when the proton
binds to it, forming a hole in the muscle cell that allows large
numbers of ions like sodium to flow in. The ions make the muscle
contract.
The researchers knew protons were being released from the gut because
they could see their effects through a microscope.
They bred worms with a green fluorescent protein that loses it color
when many protons are present. The adult worms had the green protein
in the fluid-filled space between the intestine and the surrounding
muscle – a space that Ernstrom says “is approximately 1,000 times
smaller than the width of a hair.”
The scientists were able to show protons were pumped from the
intestinal wall and to the surrounding muscle because, under a
microscope, the fluid-filled space became less green. In mutant worms
lacking the proton pump, the green remained unchanged, showing that
protons were not released from the gut into the space.
In the next experiment, the researchers added protons to the
fluid-filled space between the intestine and surrounding muscle in the
mutants lacking the proton pump. The added protons made the muscle
contract. That indicated protons indeed were acting like a
neurotransmitter to carry the signal for contraction from the
intestine to the surrounding muscle.
“To prove that it is the protons triggering the contraction, we want
to supply the protons ourselves,” says Davis. “A fine needle filled
with protons was used to inject protons in the space between the
muscle and gut. We can bypass the gut and fool the muscle into
thinking the gut is releasing protons, and the muscle contracts.”
The study was funded by the National Institutes of Health and the
Howard Hughes Medical Institute.
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