A buzzing mosquito is an all too familiar, and unwelcome, summer sound; a hungry female is searching for her next blood meal. But once she has gorged herself, she faces two major challenges: dealing with the meal's enormous volume and high salt content. Sarjeet Gill explains that although many details of the membrane transport processes involved in processing the meal have been understood for several decades, the molecular identities of the transporters have remained elusive. Knowing that sodium transport across membranes in mammals is driven by sodium/proton exchangers, Gill and his colleagues at the University of California, Riverside, decided to identify and clone the transporter (p. 3529)from the infamous mosquito: Aedes aegypti.
Basing his search on known sodium/proton exchangers from other organisms,Gill identified two key regions in these proteins that would help him to identify and locate the mosquito form of the gene. By successfully matching mRNA isolated from the insect's midgut and Malpighian tubules with these key regions, the team were able to isolate the elusive gene and clone it.
Next, the team went on to test the transporter protein's function. Cloning the new gene into sodium sensitive yeast cells that had lost sodium tolerance and all four of their own sodium transporters, the team found that the cell's sodium tolerance was restored by the mosquito gene, but only to an extent. It seemed that the mosquito transporter compensated only for the loss of the yeast's plasma membrane sodium transporter, but didn't compensate for the lost vacuolar sodium transporter.
Knowing that the transporter probably exchanges protons for sodium ions,the team decided to see if the protein restored acidity tolerance to a mammalian cell line that had lost its own sodium/proton exchanger. Cloning the new gene into the cells and exposing them to an acidic environment, the team found that the mosquito gene restored the cell's pH tolerance. Finally the team tested the mammalian cell's ability to take up radioactive sodium, and found that only the cells that had received the mosquito gene incorporated the hot sodium.
Having convinced themselves that the protein expressed by the new mosquito gene had all the characteristics of a sodium/proton exchanger, the team decided to identify locations in the insect's body where the gene is expressed. Designing an antibody that recognised a portion of the enormous transporter protein, the team probed major tissues involved in processing both fluid and ions, including the gut and Malpighian tubule, and found that the transporter protein was localised in the basal plasma membrane of the Malpighian tubule, the midgut, and regions of the gastric caeca. The team admit that they were surprised by the protein's occurrence in the Malpighian tubule's basal membrane on two counts; it is usually found in apical membranes in vertebrate kidneys, and all Malpighian tubule ion transport models developed to date have placed cation/proton exchangers in the apical membrane. The team want to follow up this discovery by finding out how the transporter is trafficked within the insect's Malpighian tubules, and to refine ion transport models, which have previously overlooked the occurrence of sodium/proton exchangers in the basal membrane.