May 01, 2003
Researchers Discover Structure of Nature’s "Circuit Breaker"
Researchers have answered an important question in biology by
discovering the exquisite mechanism by which channels in the cell
membrane sense voltage changes that trigger them to snap open or slam
shut with extraordinary speed and precision.
Voltage-dependent ion channels are central to the function of nerves
and muscles, and without them the brain would immediately suffer neural
gridlock and the heart would seize up.
“We had another strike against us in crystallizing the potassium channel protein, because it had many moving parts, which really prevented crystallization.”
Roderick MacKinnon
According to the researchers, which were led by Howard Hughes
Medical Institute investigator Roderick
MacKinnon, the discovery may lead to a new class of drugs for
neurological, heart and muscle disorders that can exert more subtle
influences on the activity of ion channels.
MacKinnon and his colleagues at The Rockefeller University published
their findings in two papers in the May 1, 2003, issue of the journal
Nature. Specifically, the researchers deduced the structure and
function of the voltage-sensing mechanism in a potassium channel of an
archaebacterium that thrives in the near-boiling temperatures of hot
springs. However, they said, the mechanism undoubtedly applies to
voltage-sensing calcium and sodium channels as well, and is present in
organisms from the most ancient bacteria to humans.
Voltage-dependent potassium ion channels are precise molecular
machines that are critical to propagating electrical impulses in the
brain and heart. The channels are large proteins with a pore that
pierces the cell membrane and is designed to allow only the passage of
potassium ions. When an electrical impulse travels along a nerve, the
charge on the cell membrane changes — with the outside becoming more
negative — triggering these ion channels to open and allowing
potassium to flow out of the cell. This outflow of potassium allows the
membrane to return to its resting state and prepare for the next
impulse.
According to MacKinnon — whose past work has revealed many new
details about the architecture and inner workings of potassium channels
— the voltage-sensing mechanism remained a frustrating mystery.
“The general principle was understood that the electric field on
the membrane somehow moved `gating charges' on the channel that would
somehow open it to allow potassium to flow,” he said.
Figuring out the location and function of the charge-carrying
structure remained a major problem, he said. “This has been a
central project for me and members of my lab for a very long
time,” said MacKinnon. “We've been working on this for
almost six years.”
Indirect experiments in other laboratories suggested that the
channel included some sort of voltage-sensing mechanism buried deep
within the protein that slid back and forth with changing membrane
charge to open or shut the channel. However, the only way to reveal the
mechanism definitively, said MacKinnon, was to obtain high-resolution
x-ray crystallographic images of the structure. Such images are
produced by beaming x-rays through purified protein crystals of the
channels and then using a computer to analyze the patterns of
diffracted x-rays to deduce the structure of the channel.
Producing the crystals necessary for such studies presented a major
challenge, said MacKinnon. “Membrane proteins are notoriously
hard to crystallize because they are embedded in this oily membrane,
and to get them out of the membrane and crystallize them, you have to
use detergents. And these detergents form a sort of `life jacket'
around the proteins, making them mushy and not amenable to
crystallizing.
“What's more, we had another strike against us in
crystallizing the potassium channel protein, because it had many moving
parts, which really prevented crystallization,” he said. After
years of testing many different potassium channel proteins and
crystallization techniques, the researchers finally found one protein
— from the thermophilic archaebacterium Aeropyrum pernix that
proved more rigid and stable when isolated. They also developed a
technique of attaching monoclonal antibodies to the
“floppy” portions of the protein, to create attachment
points for crystallization of the proteins.
The first x-ray structures using the crystals revealed a startling
surprise about the voltage-dependent potassium channel, said MacKinnon.
Instead of being embedded deep within the protein, the voltage-sensing
gating charges appeared to be incorporated in “paddles” on
the outside of the protein.
Working from the x-ray structure, the researchers theorized that
these positively charged paddles would flip-flop back and forth from
inside to outside the membrane, according to the charge across the
membrane. When the membrane became negatively charged on the outside,
the paddles would be attracted and would flip toward the outside,
opening the channel and allowing potassium to flow out, restoring the
membrane charge to its resting state. And when the inside of the
membrane became negatively charged, the paddles would flip back,
snapping the channel shut.
Indeed, in the second Nature paper, MacKinnon and his
colleagues proved that the paddles actually functioned in the way
suggested by the structure. In those biochemical experiments, they used
antibodies and other chemicals to grab the paddles on one or the other
side of the membrane, proving that they flopped back and forth with
changing membrane charge.
“So, we've shown that the membrane voltage decides whether the
channel's open, because the paddle feels the voltage and goes one way
or the other,” said MacKinnon. “It's a feedback loop. The
channel sets the membrane voltage, but the membrane voltage decides
whether the channel's open. This is precisely the kind of feedback loop
that you need in sodium and potassium channels to propagate a nerve
impulse.”
MacKinnon emphasized that, although they found the paddles in
potassium channels, the same voltage-sensing mechanism is likely to
exist in other channels. “Voltage-dependent calcium, sodium and
potassium channels are all members of the same big family and are all
related evolutionarily,” he said. “And what relates them is
that they have the same voltage sensor. So there is absolutely no
question that this is a conserved biological mechanism.”
Discovery of the voltage-sensing mechanism may yield important
clinical applications, said MacKinnon. “These paddles, I think,
will be important targets for compounds that modulate ion
channels,” he said. “Current drugs that block the ion pore
can only inhibit the channel. However, a molecule that binds to the
voltage-sensor paddle could either lock it shut or hold it open.”
Given the ubiquity of ion channels throughout the body, such drugs will
likely prove useful in a broad array of disorders of the nervous
system, muscles or heart, MacKinnon said.
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