|An Open and Shut Case
A New Look at Voltage-Gated Ion Channels in Neurons
|Contact: Lynn Yarris, [email protected]|
The neurons in the human brain are like exceptionally small high-speed transistors, controlling the flow of electrical impulses through channels in cell membranes by the opening and closing of a molecular gate. For neurons, these gated channels are specialized transmembrane proteins that form pores to let specific ions pass in and out of cells in response to a voltage change.
It is known that ion channels contain voltage-sensing domains (VSDs) and that these VSDs contain an electrically charged segment, called S4, which triggers the opening and closing of channel gates. However, there has been a great deal of scientific controversy over precisely how voltage-gating is accomplished. At stake is the possibility of new and improved drugs for the treatment of neurological disorders, migraines, and even heart conditions.
At least one part of the mystery appears to have been solved, according to the results of a study led by Ehud Isacoff, a biophysicist who holds joint appointments with Berkeley Lab's Physical Biosciences Division and the University of California at Berkeley's Department of Molecular and Cell Biology. Isacoff and Francesco Tombola, a postdoctoral fellow in Isacoff's lab, opened up a pathway for ions to go where ions have never gone before: the S4 path through a VSD. This made it possible for them to map the structural changes that take place within the VSD when voltage drives an ion channel from its electrical resting state to the activated state that opens it. Their map revealed that S4 moves inward and outward through the VSD on a tilt, while spiraling like a screw.
"This is the first model of the resting state of a VSD, and we were amazed to find the combination of a tilt and screw motion in the S4 pathway," says Isacoff. "We and others in the field had for a while thought that a screw motion could be taking place, based on theoretical considerations and experimental data, but that idea had been challenged and the field has been replete with incompatible models."
Voltage-sensing domains are supersensitive to changes in voltage across cell membranes, far more sensitive than their electrode counterparts on computer chips. In response to a voltage change VSDs undergo conformational changes that, like the opening or closing of a transistor's logic gate, either propagate or pinch off a flow of ions through the channel. X-ray crystallography images identified the VSD's S4 segment as the sensor that causes a channel's gate to open or close, and other studies showed that S4 movement involves positively charged residues (cations) of the amino acid arginine.
However, a precise model of this movement, and whether the S4 arginine cations reside in an isolated polar protein environment, or in the membrane lipid a critical factor in the design of effective pharmaceutical drugs was missing. Instead, researchers were being presented with a slew of hypothetical and all-too-often contradictory ideas.
"Some argued that S4 moves up and down in the cell membrane by about 15 angstroms, while others said the movement was no more than 1.5 angstroms," Isacoff says. "Some argued that the arginine cations lie in the lipid, while others said they lie in a polar protein pathway. These differences lead to entirely different mechanisms of voltage sensing."
For example, he explained, whereas a small S4 motion would indicate that the segment is essentially still while the membrane electric field moves across it, a large motion points to S4 itself moving through the membrane field. Realistically, for S4 to control the opening and closing of a channel's gate its motion would have to be large, which raises another conundrum.
"We wondered how in the world can arginine cations move through the membrane without going through a hole. But if they go through a hole, why doesn't the hole also leak ions from solution?" Isacoff says. "What we found, to our shock and delight, was that if you mutate one of these arginine cations into a smaller amino acid, then you do create a hole through which ions can leak. We called this the omega current."
Isacoff and his colleagues found that when S4 is in its resting conformation, the arginine cations face into a polar protein environment within the voltage-sensing domain. In response to a voltage change, S4 begins to tunnel through the membrane, creating a hole that is occupied by one arginine cation after another, which effectively blocks any other cations from leaking through. However, when smaller, electrically neutral amino acids are substituted for the arginine cations, other cations in the membrane can slip through the hole, and an omega current results. This current became stronger or weaker when mutations or biochemical modifications change the local environment so as to attract or repel the ions.
A better understanding of the structural mechanics behind ion-channel VSDs holds great promise for the treatment of damaged neurons. Current therapies are targeted at blocking ion-channel pores, but because the molecular components of these pores tend to be highly conserved by evolution, it is difficult to make channel-specific blockers. This lack of specificity yields unwanted side effects.
Says Isacoff, "There's a growing hope that far more specific inhibitors or stimulators of ion channels can be made by targeting the complex and highly differentiated structural domains of the VSDs."
Isacoff and his research group are now using an in situ optical technique he developed called "voltage clamp fluorometry" to produce dynamic images of VSDs in action. In this technique, fluorescent molecules are tagged to specific points along an ion channel. The color and brightness of the light these molecular labels emit is affected by changes in the protein structure.
"Voltage clamp fluorometry reveals local motions around a labeled site," says Isacoff. "By scanning each label through dozens of positions and measuring each individually we can reconstruct the global motions underlying voltage sensing and gating."