January 17, 2002
Images Reveal How Body Regulates Salt Uptake in Cells
Using x-ray crystallography, a team of scientists led by Howard
Hughes Medical Institute investigator Roderick
MacKinnon at The Rockefeller University has determined the
three-dimensional structure of the chloride ion channel. The images,
which were reported in the January 17, 2002, issue of the journal
Nature, reveal an entirely new type of protein architecture
designed to be an efficient conductor of chloride ions across the
membrane of cells.
The results, called a “spectacular breakthrough” by
Thomas Jentsch of the Center for Molecular Neurobiology in Hamburg,
Germany, in an accompanying News and Views article, resolve a
confusing series of biochemical studies that failed to explain how the
channel works.
“It is a complicated structure,” said MacKinnon.
“Scientists did an excellent job deducing many aspects of the
chloride ion channel. For example, its dimer architecture was predicted
20 years ago by HHMI investigator Christopher
Miller at Brandeis University, and firmly established more recently
by his laboratory using biochemical methods and electron microscopy.
But to understand the physical principles of anion selectivity, an
atomic structure is needed. Although the structure is complicated, it
conveys a simple message of how nature arranged the protein to
stabilize an anion such as chloride inside the membrane.”
Electrically charged ions are used by living organisms for many
types of signaling, including controlling the heart rhythm, generating
nerve impulses and secreting hormones. Cells use ions to signal by
creating an electrical charge difference between the inside and outside
of the cell.
The channels solve an important conundrum, said MacKinnon.
“Being charged, ions would rather be in water than in an oily
membrane. Nature has to have a mechanism to get the ion across the cell
membrane.” It accomplishes the feat through ion channels, which
are essentially pores in the cell membrane that can distinguish one
type of ion from another and only admit those that pass the selection
process.
Chloride ion channels are found throughout the animal kingdom. In
humans, nine different ClC channels regulate processes as diverse as
salt reabsorption in the kidneys and muscle contractility. Several
human disorders, including diseases of the kidney and muscle, have been
linked to chloride ion channel mutations. People with Thomsen’s
disease, for example, have chronic muscle stiffness and delayed ability
to release a handgrip. Mutations in another type of chloride ion
channel, the cystic fibrosis transmembrane regulator, cause the most
common genetic disease of people of European descent.
In 1998, MacKinnon and his colleagues solved the crystal structure
of the potassium ion channel. Potassium, which carries a positive
charge, is used for signaling in the nervous system, among other
roles.
Once this structure was discovered, the researchers set out to solve
the structure of the chloride ion channel to discern how negative ions
move across the cell membrane. They chose the ClC channel because other
scientists had shown previously that all the ClC channels, from
bacteria to humans, had the same basic protein sequence. Being able to
isolate the protein from bacteria was important because large
quantities are needed to create the high purity protein crystals
required for X-ray crystallography. The scientists chose to crystallize
the ClC protein from both Salmonella typhimurium and
Escherichia coli, two bacteria commonly studied in the
laboratory.
They discovered that the chloride ion channel has a completely
different structure from the potassium ion channel. While the potassium
ion channel has one large single pore with a water-filled,
pyramid-shaped cavity, the chloride ion channel has two pores, each
shaped like an hourglass with a narrow constriction at the center. The
scientists also discovered the arrangement of the protein subunits that
make up the channel are arranged entirely differently in the two types
of channels. In the potassium ion channel, four protein subunits
contribute to a single pore. In the chloride ion channel, each protein
subunit has its own pore and the two halves of the subunit have
opposite orientations in what’s called two-fold rotational
symmetry.
“We can see where the chloride ion binds,” said
MacKinnon. “We can pinpoint the chemistry that holds a chloride
ion and defines the selectivity filter. This is important to designing
future experiments that will test how the chloride ion flows through
the channel. By pinpointing the important chemical interactions, we
narrow down where mutations should be made to test function.”
Once the scientists saw the structure, they realized that
biochemical analysis of the protein would likely never have produced a
useful model structure.“It is complicated to figure out until you
see it,” MacKinnon said. “We needed the structure to really
understand how this protein works.” Without the x-ray structure
it would have been impossible to understand how the various parts come
together to create the pore, he added.
Now that the structure is known, said MacKinnon, it will help
scientists figure out how the channel opens and closes to maintain the
appropriate concentration of ions inside the cell. This process, called
gating, is only beginning to be understood, he said. Future experiments
in MacKinnon’s laboratory will focus on determining how the ion
channel accomplishes the gating process.
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