UCSF team reveals how the most selective of all cell gatekeepers works -- a "three-faced" channel vi

By Wallace Ravven on October 19, 2000

Advancing a 30-year quest to understand how nerve cells can precisely select
what kinds of molecules they allow in, a University of California, San
Francisco biochemist and colleagues have revealed the atom-by-atom structure of
an ancient and extremely discriminating kind of channel embedded in cell
membranes, from bacteria to humans.

The revealed structure of the cellular gatekeeper is so detailed that the
scientists have determined for the first time how the channel works, bringing
them closer to understanding how therapeutic drugs affect movement of molecules
through membrane channels—research that can help develop treatments for
kidney, brain and neuromuscular disorders.

The detailed view of this protein channel, captured by x-ray crystallography,
even shows the channel’s molecular cargo caught midway through its passage from
the outside of the cell to the inside—the first time this has been seen. The
details make possible the first demonstration of how this family of protein
channels—including those in high-powered human brains—maintain their
selectivity, says Robert M. Stroud, PhD, UCSF professor of biochemistry and
biophysics and of pharmaceutical chemistry who led the research effort.

The family of proteins is known as the aquaporins, and the ultra-selective
member of the family the researchers scrutinized through x-ray crystallography
is known as a glycerol-conducting channel.

“The whole of the human brain and nervous system, as well as the system that
provides neuromuscular control are based on highly selective membrane channels,
so to understand how these channels function shows us how the principal ‘
transistors’ of the nervous system function,” says Stroud, senior author of a
report on the research.

The article is published in the October 20 issue of Science.

The finding proves that when it comes to controlling access into the cell, size
isn’t everything. Chemistry is. The researchers were able to determine the
chemical interactions that enable the channel to allow some simple
carbohydrates free passage, yet block access to considerably smaller water
molecules and ions that provide the electrical currents of the nervous system.

The key molecule that passes through the channel is glycerol, a simple
carbohydrate that every cell employs as raw material to build and maintain the
cell membrane. Without glycerol, there would be no membrane and, without its
membrane, the cell would immediately be swamped by an incoming chemical and
electrical surge.

And yet the cell vitally needs new materials—glycerol to create its
protective barrier; ions for rapid signaling in the nervous system; and water
for cells to accommodate to the rapidly changing environment around them.
Protein channels embedded in the membrane provide needed access from the
outside, but they do so selectively. The aquaporin family of protein channels
studied by the UCSF team includes channels that let in glycerol, but not water
or ions, which are charged atoms. Other aquaporins allow in water, but exclude
ions and glycerol.

The type of channel the UCSF scientists studied has been part of the biological
makeup of organisms for least two billion years, Stroud pointed out, when
ancient single-celled organisms first needed to selectively filter what they
took in. Modern animals have inherited the genes for these channels through the
insulating cell membrane, and have adapted them for regulated movement, or
conductance, of different molecules. Today the glycerol-conducting channels are
found in bacteria such as E. coli, in yeast, plants, and all animals, including
humans. They are vital to normal function of the human brain, eye, kidney and
other internal organs.

The researchers focused on the most selective of these channels. They
crystallized a glycerol-conducting channel from E. coli and analyzed the
structure in atomic-level detail using x-ray crystallography.

The images of the crystal structure actually caught the glycerol molecule in
the process of passing through the membrane’s protein channel. This “midstream”
capture had never been accomplished before. The stop-action image and knowledge
of the chemical interactions between glycerol and the molecules that make up
the inner surface of the membrane channel, allowed scientists to determine that
glycerol is essentially a three-faced molecule—physically and chemically.
The glycerol channel, too, has three faces, each of which interacts with the
glycerol molecule to selectively filter only this molecule into the cell.

To pass through the “three-faced” channel, a molecule must also have three
aspects: a “water-hating” side, a hydrogen-donating side (an oxygen atom with
attached hydrogen), and a hydrogen-accepting side (an oxygen atom) on the its
third face. Glycerol fits these conditions and is filtered through the channel
into the cell.

Water, on the other hand, though a smaller molecule, could only fit through the
channel opening in single-file—each water molecule bound only to the one in
front of it and the one behind it in the queue. It is energetically costly for
water molecules to strip off their bonds to other water molecules, Stroud
explained, and so water can not pass through the channel. Similarly, ions, that
normally must be bound to water for safe passage, can not get through the
channel.

By defining precisely how the channel can conduct glycerol into the cell
without passing any charge-containing ions that might leak away the cell’s
“batteries” of stored energy, Stroud’s group showed in the greatest detail yet
precisely how trans-membrane channels work.

X-ray crystallography, the method used to provide the atomic-level view of the
membrane channel’s molecular structure, works by detecting the orbital patterns
of electrons. The approach relies on a “light” source with wavelengths close to
the distance between atoms in the crystal being studied. Stroud’s group uses
computer processing of diffracted x-rays to “see” the exact positions of atoms
in the crystal structure.

Lead author on the Science article is Daxiong Fu, PhD, postdoctoral scholar in
Stroud’s laboratory. Co-authors, all in Stroud’s lab at UCSF, are Andrew
Lisbon, PhD; Cindy Weitzman, PhD; and Peter Nollert, PhD, all postdoctoral
scholars; and Larry Miercke, MS, and Jolanta Krucinski, MS, research
specialists.

The research was funded by the National Institutes of Health.