Channelling a path for substrates through a multidrug transporter

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Understanding nature's "bouncers": the mechanism of multidrug pumpsAll cells are surrounded by a protective barrier known as the cell membrane. Cells control what they transport across this membrane, in order to be able to take up (import) or remove (export) specific chemicals. Examples of important transport include taking up nutrients from the diet into the cells lining the gut, export of toxins and waste chemicals into urine and bile, and secretion of vitamins and nutrients into milk in mammary tissue. These transport processes are carried out by "pump proteins" in the cell membrane. Pump proteins usually transport a specific molecule in a set direction (i.e. either into the cell or out of the cell). Some pump proteins are unusual in that they export from cells not just one type of chemical, but hundreds of different and unrelated chemicals. These proteins are called "multidrug (MDR) pumps" and they are the cell's equivalent of a nightclub bouncer, with actions that can have a big impact. For example, antibiotic resistance in bacteria - a huge healthcare challenge - can occur because multidrug pumps remove antibiotics from their target cells. In humans, MDR pumps protect cancer cells from chemotherapy because the pumps are expressed more in tumour cells. They kick the anticancer drugs designed to kill tumours back out of cells and cause chemotherapy to fail. These MDR pumps can also affect how we all respond to medication for a wide range of conditions including statins for heart disease and anticonvulsants for epilepsy. MDR pumps in the gut, liver and kidney control how these medicines are absorbed and removed from the body. MDR pumps are not always "bad news" - the chemicals industry wants to use bacteria to make chemicals (such as fuels) from simple sugars, rather than using dwindling fossil fuels. MDR pumps are important in this "green chemistry", because they can be used to export the useful chemical products from bacteria for harvesting. A deep understanding of how MDR pumps work, and how we can hijack or block this process, is therefore really important. In other pump proteins that transport only one chemical, the route this chemical takes through the protein as it is 'pumped' can be well defined. However, one of the main challenges in understanding MDR pumps is that these can deal with so many different drugs and chemicals so mapping their route through the pump protein is very difficult. We are going to tackle this big question for one MDR pump, ABCG2, in our proposal. What makes our research unique is that we have developed a new way to "see" how ABCG2 recognises chemical substrates at a microscopic level. We will use molecular "cookie cutters" to make tiny rings of cell membrane, each containing just one pump protein. We then watch a fluorescent drug (that glows when we shine laser light on it) bind our membrane "cookies" containing ABCG2. This tells us how well our substrates recognise the pump as it works, and how other drugs might stop this process. We can test our substrate route map by changing parts of the pump protein involved to see what happens to its function, and using computer modelling to simulate the interactions. We will also look for novel drug types that bind ABCG2 and regulate function, expecting that these will be useful experimental tools and starting points for future medicines.The techniques will be adapted to look at any pump protein interacting with its substrate, which will excite people interested in the fundamental roles of pump proteins in all aspects of physiology. We are already discussing with pharmaceutical and chemical production companies how a better understanding of pump:substrate interactions benefits the drug design process and chemicals manufacturing. This research project will therefore advance understanding of the basic biochemistry of an important human MDR pump, and cast a broader light on the large family of membrane pump proteins.