Unravelling the function of the Nha gene family

Around a third of our genome (indeed, a third of any organism's genome) is devoted to genes that mediate transport across membranes. Transport is essential to survival, as evidenced by genetic diseases caused by defective transport genes; cystic fibrosis is a particularly prominent example in Celtic countries. Given the importance of transport, it is surprising how many genes still need function ascribed to them experimentally. Recently, a new class of exchanger gene called CPA2 has been identified. It can be found from bacteria to humans, and exchanges sodium ions for hydrogen ions. One of the two human genes has already been identified as a candidate in essential hypertension, and in bone resorption - two processes central to human ageing. It is thus important to find out what the CPA2 gene family does in higher organisms. We plan to study the CPA2 genes in an established genetic model, the fruit fly Drosophila melanogaster where (as in humans) there are two CPA2 family members. Using Drosophila confers several advantages: the work is quicker (it takes only three months to generate a transgenic fly), cheaper ( it takes only tens of pounds to keep a line of flies alive for a year), and ethical (no experiments on mammals are required). We thus see work in Drosophila as an exciting adjunct to continuing medical study of humans, and the likely generation of mouse models in other labs. Our work will focus on identifying what the genes do, particularly in the fly 'kidney' where we have recently shown that the genes are strongly expressed. We will study the transport properties of the protein in the test tube, and use the information to explain what they may contribute to the functioning of the whole organism. Insects are important in their own right, as over a million lives are lost to malaria alone each year; and the WHO claim that a third of world crops are lost to insect attack in the field or in storage. We plan to extend our results to close relatives of Drosophila, the mosquitoes, in order to see whether the results we have generated are applicable across the insects. If so, the CPA2 family may be targets for novel insecticides.

Recently, a new class of exchanger gene, called CPA2, has been identified. It can be found from bacteria to humans, and exchanges sodium ions for hydrogen ions. By contrast with the traditional NHE (CPA1) family of electroneutral exchangers, the CPA2 exchangers are electrogenic. One of the two human genes has already been identified as a candidate in essential hypertension, and in bone resorption - two processes central to human ageing. It is thus important to find out what the CPA2 gene family does in higher organisms. We plan to study the CPA2s both in vitro and in the fruit fly Drosophila melanogaster where (as in humans) there are two CPA2 family members. Using Drosophila confers several advantages: the work is quicker (it takes only three months to generate a transgenic fly), cheaper (it takes only tens of pounds to keep a line of flies alive for a year), and ethical (no experiments on mammals are required). We already have data to suggest that both fly CPA2s co localize with a plasma membrane V-ATPase in the fly renal (Malpighian) tubule, and that the two exchangers may have differing preferences for sodium and potassium. The major objectives of the work-plan are to: - measure each CPA2 member across the major tissues, to test the model that they are partners for the plasma-membrane V-ATPase - reconstitute each CPA2 member in purified proteoliposomes, in order to determine their preference for Na/H, K/H, or Na/Li exchange - identify inhibitors for the CPA2 family in animals - test the role of the exchangers by generating single and double RNAi knockdown flies. - extend the work to the closely related Diptera, the malarial and yellow fever mosquitoes. Together, the work-plan should provide a rapid, clear and multifaceted understanding of the roles of this new gene family in a metazoan.