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A detailed view of a voltage-dependent potassium ion channel has been revealed by US researchers using XRD. The structure shows the channel in a more natural state, revealing how attendant lipid molecules within the cell membrane influence channel function. Membrane-bound proteins, such as the voltage-dependent potassium ion channels among the most fascinating molecules in biology involved in the function of nerves and muscles and countless other vital processes. However, they are notoriously difficult to crystallise and even if that is possible the structures obtained are not necessarily close to the shape of the functioning protein. Now, Roderick MacKinnon and colleagues at the Howard Hughes Medical Institute at Rockefeller University, New York, have developed a new technique, lipid-detergent-mediated purification and crystallization. MacKinnon told SpectroscopyNOW that it was impossible even to purify the protein in a stable state without the presence of lipids. The development of this new approach could now open the door to studying the hundreds of membrane proteins previously inaccessible in their natural environment to crystallography. Previously, MacKinnon and his colleagues revealed how changes in membrane electrical polarity are involved in the opening and closing of voltage-dependent potassium ion channels. Crystallography uncovered the structure of the voltage sensor essential to this process. A picture of a helix-turn-helix structure in the voltage sensor emerged. This voltage sensor paddle contains positively charged amino acids that allow the structure to respond to the membrane's electrical polarity. An understanding of how this paddle actually moves within the membrane at the protein-lipid interface remained elusive, although MacKinnon's team had their theories. They reasoned that when the interior of the membrane becomes positively charged, the paddle moves towards the outside and opens the channel. This lets potassium ions flood out, which in turn returns the membrane charge to its resting state. Conversely, when the inside of the membrane is negatively charged, the paddles move inward closing the channel and stemming the flow of potassium ions. However, several questions remained unanswered. "We could not see many of the individual side chains of this protein that are important to its function," MacKinnon said. A new approach to capturing the active structure was needed. "These are very difficult structures to determine, and our progress has been like taking one step at a time up a very big mountain," MacKinnon adds. The breakthrough step up that mountain was to engineer a new form of the ion channel that would succumb more readily to crystallisation and so provide higher quality X-ray data. The researchers produced a ?paddle-chimera? channel by swapping the normal paddles of a channel with those from a different channel. This led to a new crystal packing pattern that helped the team pinpoint those atoms in the protein that were ambiguous in the original structure. Additionally, they also capture the structure of the cell membrane itself. This simply involved immersing the channel protein in a mixture of detergent and lipid, rather than detergent alone, in the crystallisation process, although arriving at the optimum ingredients and concentrations was not simple. "This new approach gave us dramatic new insight, because we could actually see the lipid molecules gathered around the protein, and see them form the characteristic leaflets of the bilayer biological membrane," MacKinnon explains, "With an earlier structure that we published in 2005 we could only speculate why the use of lipids was important, but now we can see it very clearly," he adds. Having this more naturalistic structure to hand has changed MacKinnon's perspective on the precise role of the membrane in ion channel function. "I used to think that the voltage sensor didn't have much to do with the lipid membrane," he explains, "But these structures have informed us that the voltage sensor has a great deal to do with the lipid membrane." He points out that the "Mickey Mouse ears" of the voltage sensors sticking out must be influenced by the lipids that so obviously surround them. "This influence is so profound," MacKinnon adds, "that you can't simply say what the properties of a given voltage-dependent channel are without specifying the composition of the surrounding lipid." What is most intriguing about this discovery is that different cells in the body have different lipid composition. This implies that the function of voltage-dependent channels could vary considerably in different cells depending on lipid environment. The next step is to investigate the channels in those different environments. Related links: The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.
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