Cell's gatekeeper for ammonia revealed in unprecedented detail

Bacteria thrive on it, red blood cells carry it in high concentration, yet the human brain can’t tolerate it. The ability of cells to acquire or dispose of ammonia can be a matter of life and death. In the September 10 issue of Science, UCSF researchers publish the first atomic-level structure of the channel that controls the passage of ammonia in and out of cells.

The complex structure—depicted on the cover of Science—was determined in greater detail than that of any cell membrane channel yet described. The structure can help reveal the molecular basis of often life-threatening diseases caused by ammonia toxicity, and provides the “template for designing drugs to treat the diseases,” said Robert Stroud, PhD, professor of biochemistry and biophysics and of pharmaceutical chemistry at UCSF and senior author of the paper in Science.

Knowledge of the channel architecture is also expected to help explain how ammonia, carbon dioxide and other gases are taken in, transported between cells and excreted, the scientists said.

The issue of Science also includes a “Perspective” article about the significance of determining the structure and its likely mode of action.

The three-dimensional shape of other cell membrane channels—those that conduct water and charged molecules called ions—have been reported over the past six years. In 2000, Stroud’s group was the first to discover the structure and mechanisms of the water-conducting channels.

To determine the structure of an ammonia channel, the UCSF scientists focused on AmtB, a protein that facilitates ammonia uptake in bacteria. The bacterial AmtB is a member of a “superfamily” of protein channels with very similar amino acid sequences and very similar functions in all species. Humans have four members of this superfamily of cell membrane channels. They are the Rh factors, most familiar as the Rh blood group antigens on the surface of red blood cells that can cause the potentially lethal antigenic incompatibility of mother and child.

Rh antigens have only recently been found to be transmembrane channels that regulate the movement of ammonia and carbon dioxide in and out of the red blood cells, Stroud said.  The red blood cells transport ammonia from tissues to the kidney, and transport carbon dioxide to the lungs. The gases are excreted from these organs.

“Knowing the mode of action of Rh factors, we now know the basis of ammonia transport from tissues to the kidney, and the molecular-level mechanism for ammonia excretion via the kidney,” Stroud explained.

The structural basis for ammonia transport by AmtB is essentially the same for the human Rh factors, where small differences in the amino acid sequences of the conducting machinery lead to slight differences in their channels and provide for regulation and localization of the ammonia transport, Stroud said.

Using X-ray crystallography, the researchers determined the channel’s structure down to an unprecedented resolution of 1.35 angstroms, or just over a 100-millionth of a centimeter, they reported. In the technique, proteins are crystallized and then exposed to intense X-ray radiation. The pattern created when the X-rays are scattered off electrons in the crystal can be used to visualize the relative three dimensional position of every atom in the protein’s structure.

The research reveals that AmtB channels cluster in groups of three. Three slightly different proteins are known to associate in the blood group antigenic Rh factors, Stroud said, and the new research now reveals how they are physically associated.

While some ammonia can pass through cell membranes without any assistance from a protein channel, the ammonia channel is extraordinarily efficient at transporting ammonia—a trait called “high affinity” that can be critical since the ammonia is highly toxic, Stroud said. Many neurological disorders, from seizures to stupor and coma, stem from overexposure to ammonia. Advanced liver failure disrupts central nervous system function when ammonium ion concentrations are elevated, research shows, so the channel’s high efficiency is vital.

Each channel threads in and out of the cell membrane 11 times and is structured so that no ammonium ion, NH4+, nor any other ion, can pass through. As NH4+ enters the channel from either end—either moving from outside the cell to the interior or in the reverse direction—a proton is stripped away, leaving ammonia gas, NH3. When the ammonia reaches the other side, it regains a hydrogen atom again to become NH4+.

“This mechanism provides the precise filter that prevents any other molecules or charged molecules from leaking through this channel,” Stroud said. “This ensures the health of the cell while providing a way for the cell to transport nitrogen in this form. Ultimately nitrogen is essential for building proteins and other processes.” Bacteria and yeast, he pointed out, can survive with ammonia as their sole source of nitrogen.

The lead author on the SCIENCE paper is Shahram Khademi, PhD, a postdoctoral scholar in Stroud’s lab. Co-authors are research assistants Joseph O’Connell III, MS; Jonathan Remis, BS; Yaneth Robles-Colmenares, BS; and Larry J. W. Miercke, MS, a research specialist, all in biochemistry and biophysics at UCSF.  Stroud’s lab is part of the California Institute for Quantitative Biomedical Research, or QB3, headquartered at UCSF’s Mission Bay campus.

The research was supported by the National Institutes of Health.

NOTE: An atomic-scale image of the ammonia channel, showing the passage of NH4+ and NH3 through the channel, can be downloaded at: Atomic-scale image of the ammonia channel