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How a cellular pore opens and closes
It turns out that the cystic fibrosis chloride channel, which allows salts in and out of cells throughout the body, is structured like a peanut butter sandwich
BY WHITNEY CLAVIN
For 10 years, David Gadsby has been essentially blindfolded as he tried to open a cellular door secured by both key and combination locks.
Recently, he succeeded in unlocking the door, and revealed the workings of the cystic fibrosis chloride channel, one of the specialized openings in the membrane of cells and the protein that is defective in people with cystic fibrosis.
The cystic fibrosis chloride channel’s primary job is to control the flow of water into and out of cells, a process that is critical to maintaining the appropriate salt/water balance in the cells of people both with and without cystic fibrosis. When this balance is disturbed, as happens in cystic fibrosis patients, who don’t have properly functioning chloride channels, thick mucus accumulates in the lungs, forming a breeding ground for life-threatening bacterial infections.
Gadsby’s findings, which were reported in a series of three recent papers published in the Journal of General Physiology, unravel the molecular steps needed to open and close this channel, letting salt ions in and out of the body’s cells.
The Rockefeller scientists’ findings, generated using a combination of biochemistry and electrophysiology techniques, show that the channel is less complicated than previously believed. “Once we solved the uncertainties of the old preliminary model, a new simplified version emerged,” says Gadsby, who is head of the Laboratory of Cardiac/Membrane Physiology.
There are two major types of ion channels in cell membranes. Voltage-gated channels — such as the potassium ion channel that was structurally solved this year by Rockefeller Nobel Prize-winner Roderick MacKinnon — are triggered to open and shut by electric signals. Ligand-gated channels, in contrast, require a special key, which usually takes the form of a small molecule.
The chloride channel studied by Gadsby is a ligand-gated channel, but it’s an unusual one. Its key is ATP, a small molecule that plays a critical role in the storage and release of energy within cells in the body. Based on previous research dating back to 1994, Gadsby had a preliminary model of how ATP interacted with two separate sites near the entrance to the channel’s pore. These sites, called NBD1 and NBD2, were thought to mediate opening and stabilization of the channel pore.
The team used a tool known as a patch-clamp, in which an exquisitely sensitive electrode is placed on the cell membrane so researchers can measure the tiny electrical signals generated by the opening and closing of individual channels.  The pattern of the signals helped Gadsby and his Rockefeller colleagues learn how the channels work.
The studies revealed that the channels work in the following manner: First, ATP molecules must be bound at both NBD1 and NBD2 to cause the channel to open. It does not then close until the ATP bound at NBD2 is snipped. Additional biochemical studies demonstrated that the ATP molecule at NBD1 often stays attached while ATP comes and goes at NBD2, each time causing the channel to open and close.
To understand the basics of the channel’s opening and closing mechanism, visualize a peanut butter sandwich and think of the peanut butter as the two ATP molecules. As long as both ATPs are in place, the slices of bread — NBD1 and NBD2 — stick together, and the channel is held open. When part of an ATP is snipped off, the sandwich can no longer hold itself together and so the channel snaps closed.
Ultimately, the research may have medical applications, though, ironically, not for most cystic fibrosis patients. For about two-thirds of these individuals, the body’s cells don’t have any cystic fibrosis chloride channels, so a cure for them is expected to result from research focused on replacing the lost channel, not on studies of its mechanism.
The research has more concrete implications for diseases involving other members of the family of proteins to which the cystic fibrosis chloride channel belongs, such as the sulfonylurea receptors associated with hyperinsulemic hypoglycemia and the multidrug resistance proteins that interfere with cancer treatments.
“Understanding the molecular mechanisms of the cystic fibrosis chloride channel might help us understand how other members of the family function,” says Rockefeller University Research Associate Paola Vergani, a co-author of two of the papers.


December 12, 2003



 

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