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of the Ion Channel
 

 

 

 

 

 

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by Neeraja Sankaran  

Professor Roderick MacKinnon set up his Laboratory of Molecular Neurobiology and Biophysics at Rockefeller with a specific goal in mind—to obtain a high-resolution three-dimensional structure of a potassium ion channel protein. He fully expected that his quest for the structure would be a long-term undertaking, requiring at least a few years. But less than two years after his arrival here, MacKinnon has already accomplished his mission, as evidenced by the image of the X-ray crystallographic structure of an intact potassium channel protein on the cover of the April 3, 1998 issue of Science.

MacKinnon, who was appointed an investigator with the Howard Hughes Medical Institute last year, modestly attributes his success to "some very lucky breaks and extremely hard work on the part of my entire laboratory."

Outside experts, however, are far more forthcoming with their praise for this achievement."A remarkable accomplishment," proclaimed Clay Armstrong of the University of Pennsylvania, who reviewed MacKinnon’s paper in the same issue ofScience. "It is a dream come true for biophysicists."

Ever since their discovery in the 1950s, ion channels have been the subject of intense interest to many scientists because of the key role they play in maintaining some of the body’s most vital functions. Seated in the oily layers of the cell membrane that preserves the integrity of each cell, these proteins govern the flow of different ions such as potassium, sodium and calcium into and out of cells. The proper balance of these ions is essential for fundamental operations such as the transmission of nerve impulses throughout the body and brain.

Shaped like tiny doughnuts floating in oil, the ion channels perform the dual functions of gateway and gatekeeper. The holes in the doughnut form the gateway through which the ions flow. However, these holes, or pores, are endowed with special properties that enable different channel proteins to be selective as to which ions they allow passage. Like security guards who check ID before allowing entrance, these molecular gatekeepers only allow specific ions to pass through their pores.

What allows the ion channel proteins to work as they do? This is the question that has engaged MacKinnon for over a decade, beginning with his observation of an experiment monitoring the electrical activity of a potassium channel, when he was a medical student just contemplating a switch to basic science. He joined the laboratory of his undergraduate mentor Christopher Miller at Brandeis University, where, as a postdoc, he began to work on biophysical aspects of channel function. He chose to focus specifically on the protein selective for potassium ions, because it happened to be the target of Miller’s lab and also because, at the time, it was the least-studied ion channel, says MacKinnon.

Beginning with electrophysiological and biochemical approaches, MacKinnon studied the interaction of the potassium channel with a specific toxin derived from scorpion venom and deduced that the toxin inhibited the flow of ions by sitting directly on the pore of the channel. This led to questions about which specific regions of the channel proteins bind to ions and the toxins. Investigations along those lines became possible after 1987, when scientists cloned the genes for potassium channels in fruit flies.

In the wake of this development, MacKinnon turned to the field of molecular biology. By systematically mutating the channel gene at specific locations and observing the effects on the channel’s ability to bind to the specific ions, he was able to pinpoint ion-binding capacity to a single region of the protein.

"All potassium channel proteins contain a little signature stretch of about 8 to 10 amino acids, which is specific for the ion," explains Mackinnon. "The sequence is conserved across evolution from bacteria to humans. In fact, some of these proteins have no features in common except for this signature. It’s as if biology chose only one way to select for potassium ions."

Meanwhile, genetic evidence suggested that a single potassium channel consisted of multiple protein subunits. MacKinnon determined that a functional channel has four identical subunits that join together like the staves of a barrel around the central hole. Each of the subunits contains the ion-specific signature sequence, "which forms a loop extending into the center of the hole to create a pore selective for potassium ions," he explains.

The pore-forming loops seemed to govern specific functions such as ion selectivity, but without a clear idea of the 3-D structure of the channel, MacKinnon says there was no way to test the truth of this idea. So he came to Rockefeller where he hoped to master crystallographic techniques and solve the structure of the potassium channel.

The very nature of the problem led MacKinnon and others to anticipate slow progress in their work. A basic requirement for protein crystallography is that scientists have sufficient quantities of the starting material or protein, in order to grow crystals for X-ray analysis. But although potassium channels are present in virtually all cells, they are usually produced in low quantities.

Fortunately, this hurdle was overcome when a group of scientists discovered and cloned a potassium channel from the bacterium Streptomyces lividans, using the very signature sequence that MacKinnon had discovered a few years earlier.

"The bacterial system cleared the path by giving us a way to express channel proteins in much larger quantities than had been possible," remarks MacKinnon.

Armed with sufficient amounts of channel protein, the scientists could turn their attention to tackling their other problem, namely growing good crystals. Historically, membrane-bound proteins like ion channels have been notoriously bad candidates for protein crystallography, because the detergents used to separate such proteins from lipids—a necessary step in making good protein crystals—often destroy the proteins as well.

"To give an idea of the labor involved in determining the ideal parameters for growing good crystals, we concocted a crystal screen with a total of 900 different conditions at two different temperatures and with 10 detergents," says MacKinnon. "Luckily we hit on the right combination relatively early."

The structure of the potassium channel (see figure) confirms MacKinnon’s theory about the pore–loop structure determining ion selectivity and also offers an explanation as to why the channel prefers potassium over sodium ions. "Basically the potassium selectivity filter is a cylindrical cavity lined by slightly charged oxygen atoms which, in turn, are held in place by structural elements in the portion of the channel protein that traverses the membrane," explains MacKinnon.

This structure confers a certain rigidity to the cylinder and determines the diameter of the oxygen-lined pore. A charged potassium ion fits neatly into the narrowest configuration of this cylinder. Sodium ions, which are smaller, do not fit properly in the cavity. "The oxygens in the pore cannot get as close to the sodium as they do to potassium. Consequently sodium ions are better stabilized by oxygens in water molecules and do not enter the pore," MacKinnon adds.

The channel structure also suggests an explanation for the seeming paradox of a channel protein’s ability to reconcile high ion selectivity with high throughput, or the rapid flow of ions through the channel.

"The two properties seem incompatible because high selectivity implies that the interaction between the channel and ion must be a strong one, which would not be conducive to letting the ions escape past the protein into the cell," MacKinnon says.

But it appears as though the selectivity pore resolves this paradox by placing two ions near each other where they experience mutual repulsion by virtue of their electric charge. "This suggests that the strong attractive forces between the potassium ions and channel are counteracted locally by the repulsive forces between two positively charged potassium ions, which allows each ion to fall through at a rapid rate," he adds.

Having solved the elusive structure of the channel, MacKinnon plans to obtain information about channel structure at still higher resolutions and probe other aspects of their function. Ultimately, he says, "My aim is to figure out how channel proteins function as the electrical impulse generators in biology."

This article originally appeared in the Spring 1998 issue of Search, a Rockefeller University publication.

 
"My aim is to figure out how channel proteins function as the electrical impulse generators in biology."
 
 

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