Potassium ion channels are responsible for the rapid, selective flow of potassium (K+) ions through cell membranes, making it possible for spikes of electric activity (action potentials) to rapidly travel along membranes to coordinate the action of our hearts, brains, and muscle. But how does that work? About 25 years ago, Rod MacKinnon established the structure of these channels using cryogenic X-ray crystallography. This structure revealed K+ ions sitting in a narrow region, the selectivity filter, in which they are stripped from their surrounding water molecules and are instead surrounded by four consecutive “cages,” each consisting of eight oxygen atoms of the protein. Because Na+ and K+ ions differ in size, they have different energies when surrounded by six or eight oxygen atoms both in water and in the selectivity filter, making it possible for the channel to let through only K+.
This was a major milestone. Yet it left important questions open: How many K+ ions are there in the selectivity filter? Does the passage of ions look the same in both directions? Are the motions of the channel coupled to those of the ions passing through? If so, could this tell us anything about the evolutionary success of these channels?
Since the first ion channels were crystallized, scientists have wanted to directly observe the movement of ions through the channel. MCB faculty Doeke Hekstra , with collaborators at Argonne National Lab, Stanford, and the University of Chicago, began pursuing this goal after developing electric field-stimulated time-resolved X-ray crystallography, or EF-X for short. In an EF-X experiment, electric field pulses are applied to protein crystals using electrodes, while the crystal is simultaneously exposed using X-ray pulses to record the changes in the structure within the crystal.
“Before turning on the electric field, we first established that–at room temperature–the selectivity filter is almost fully occupied by K+ ions in our model ion channel —unlike in early models of ion permeation but consistent with results obtained under cryogenic conditions more recently,” Hekstra says. This observation was very helpful in interpreting our results: before we turn on the electric field, there is a single initial pattern of where the ions are rather than some complicated mixture.”
Hekstra continues, “With EF-X, we can now see these dynamics directly: to see ions come and go. Indeed, we can see the departing ions put on a new “coat” of water on their way out. To our surprise, the intermediate states looked quite distinct depending on which direction the ions flowed. This may be explained by another observation: the channel slightly deforms, like the twisting of an iris in a camera, with the direction of the twist depending on the direction of ion flow.”
It took quite a few steps to learn how to perform these experiments effectively: “We produced a graveyard full of prototypes going by names such as the ‘pizza saver’, the ‘bath tub’, and the ‘globe holder,’” Hekstra says. “The key to success turned out to be a complete redesign of the electrodes and protocol, simplifying the treatment of sensitive crystals like those of the K+ ion channel. The benefit of working with such a delicate target—a membrane protein—is that we now have an approach in place that should work for a large variety of proteins, rather than a few exceptional ones accessible in the first EF-X experiments.”
Indeed, these experiments were the first ones to take advantage of a permanent setup supporting EF-X experiments that Hekstra designed in close collaboration with Dr. Robert Henning the BioCARS facility at the Advanced Photon Source at Argonne. The setup is available to anyone when the Advanced Photon Source reopens this Winter after billion-dollar upgrade of its electron ring. We can’t wait to see what will be possible with this new beam!
Hekstra Lab Website
