Exploitation of a Pharmacological 'Master Switch' to Probe the K+ Channel Selectivity Filter Gating Mechanism

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Almost every living cell is electrically active because a voltage difference exists across its membrane. However, unlike the electronic currents we are so familiar with domestically , this 'biological electricity' results from the movement of charged ions (like Na+ and K+) into and out of cells through tiny pores in their membrane known as 'ion channels'.These ion movements generate electrical currents that control or influence almost every process in the human body, from the way we move and breathe, the way our heart and kidneys work, to the way we think, and how we sense pain. Even bacteria use similar changes in their membrane potential to control important processes. So it is no overstatement to say that ion channels are essential for all forms of life on earth. As a consequence, a wide range of medicinal drugs and many poisons exert their effects by regulating the activity of these tiny molecular machines, and many human and animal diseases result from impaired ion channel function.Potassium (K+) selective channels are one of the largest groups of ion channels; there are >80 different human K+ channel genes and K+ channels are found in almost every living organism (and even some viruses). As a result, their structural architecture is very highly conserved across these different life forms.To operate effectively, all K+ channels need two key properties: a 'filter' so they can be selective for K+ over other ions, and a 'gate' that opens and closes to regulate the flow of ions. This opening and closing or 'gating' is regulated by many different cellular signals (including many potential drugs). This makes K+ channels attractive therapeutic targets for the treatment of disease.The first 3D structure of a K+ channel was determined exactly 20 years ago and revealed how the K+ selective filter and gate are formed by two distinct features at either end of the channel. However, more recent studies now suggest that in some types of K+ channel the normal (lower) gate remains open, and instead all regulation of ionic flow is done via the selectivity filter which acts as both filter AND gate.Unfortunately, the mechanism of filter gating has been difficult to study because few tools exist to manipulate this process. Some computational approaches can directly study the permeation of individual ions, but these measurements are often limited to just a few millionths of a second and even the most sensitive of electrical recordings are unable to directly measure ion channel activity on this timescale. Consequently, it is difficult to relate the movement of ions to the 3D structure of a channel and new approaches to this problem are required.Luckily, we have now identified a new class of drugs which interact with the filter gate to directly activate the flow of K+. Interestingly, these drugs work across a range of different K+ channels and therefore provide a new set of tools to manipulate the filter gate and dissect its mechanism of action.In this project we propose to exploit these exciting new tools to manipulate ion permeation through single ion channels. Other approaches will also allow us to compare our measurements with structural and computational models of permeation. In addition, these studies may allow us to classify (and predict) how other K+ channel drugs interact with the channel, and where they may bind. This will have important implications for the rational design of new drugs that target these channels.Overall, this proposal is supported by extensive data that demonstrate the feasibility of our approach. It therefore offers a unique and timely opportunity to understand this most fundamental of biological signalling processes, and it will improve our ability to design new drugs that target K+ channels.