Oct. 24, 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.
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