Ion transport at the blood brain barrier, pH regulation, multidrug-resistance transporters in red blood cells and chloroquine accumulation by malaria parasites (Plasmodium falciparum).
Current work is centred on three themes:
- characterisation of ion transport in brain endothelial cells and its relation to the normal secretion of brain interstitial fluid and to the formation and resolution of brain oedema
- the function and roles of mutlidrug-resistance transporters in red blood cells
- chloroquine uptake by the malaria parasite, Plasmodium falciparum
In a collaboration with Dr. Margery Barrand, we aim to determine the mechanism of fluid secretion at the blood-brain barrier and how this process is altered following hypoxia/reperfusion. Traumatic brain injury, inflammation and cerebral hypoxia/reperfusion can all lead to impairment of brain function. While after a stroke there is a core region in which damage is immediately irreversible, a wider region only becomes irreversibly damaged over a period of time. One contributor to this spread is the formation of oedema (brain tissue swelling) that occurs because the rate of production of brain interstitial fluid exceeds the rate of its removal. In the absence of gross breakdown of the blood-brain barrier, the activities of transporters, i.e. exchangers, channels and pumps, determine the rate of production of brain interstitial fluid. We expect that understanding the basic secretion process will provide pointers to suitable additional therapeutic targets for limiting the spread of the region of irreversible damage that occurs in the critical hours following a hypoxic episode.
Our work approaches this subject using a combination of techniques: real-time PCR and immunocytochemistry to identify and locate the transporters present; fluorescent indicator measurements and confocal cell imaging to follow the changes in intracellular composition and volume that couple the transport events on the two sides of the endothelial cells; patch clamp recording to characterise the ion channels present and radiotracer flux measurements to monitor fluxes. The patch clamp recording is done in Manchester in collaboration with Dr. Peter Brown. We now know the principal components of the system but much remains to be done to describe how they interact to produce the secretion at a controlled rate and with a regulated composition.
In a second collaboration with Margery Barrand we have been investigating the functions of multidrug resistance associated proteins (MRPs) in red blood cells and malaria parasties. The MRPs are examples of ATP binding cassette (ABC) transporters. MRP1 is found in the membranes of a number of malignant cells that display resistance to many, seemingly unrelated, chemotherapeutic agents. In these cells it serves to expel the drugs thus thwarting treatment. However, it and its isoforms are also present in many normal tissues including the liver, the linings of the lung, and red blood cells. We have shown that in red cells MRP1 is the high affinity transporter for oxidised glutathione (GSSG) while the low affinity GSSG transproter, which also appears to export cyclic nucleotides, resembles MRP4. We have also provided preliminary evidence of the presence of MRP-like protein in the most lethal form of malaria parasite, Plasmodium falciparum.
The third theme for my research, the uptake of chloroquine by Plasmodium falciparum, is a collaboration with Prof. Kiaran Kirk (A.N.U., Canberra) and Prof. Leann Tilley and Dr. Nick Klonis (Latrobe University, Melbourne). For almost 40 years chloroquine was a miracle drug (cheap and effective!) for the treatment and prevention of malaria attacks which spawned the hope that malaria might be conquered. However, instead over the last 20 years Plasmodium falciparum, the causative agent for the most lethal form of malaria has developed resistance to chloroquine and at present is responsible for about 2 million fatalities per year. Chloroquine resistance is relative, the parasites can still be killed by chloroquine, but only at concentrations about 10-30x higher than before, concentrations so high that they are harmful to the infected person. Reversal of this resistance would have truly dramatic consequences.
It has long been known that chloroquine-sensitive P. falciparum within the red blood cells of an infected person accumulates large amounts of chloroquine in its acidic intracellular digestive vacuole and that chloroquine binds strongly to the products of the haemoglobin digestion. Chloroquine-resistant parasites accumulate 10-30x less. Both the clinical resistance and the deficit in accumulation have been traced to a single mutation in the gene for a protein, PfCRT, that is located in the membranes of the acidic compartment. We have constructed a mathematical simulation of chloroquine accumulation in P. falciparum which for the first time takes into account both the pH and potential dependent distribution of free chloroquine within the parasites (so-called weak base trapping of the charged forms of chloroquine) and a simplified description of the binding of chloroquine as an equilibrium binding of the diprotonated form of chloroquine to the form of haem released from the haemoglobin. The role of the mutated PfCRT in these simulations is to allow protonated chloroquine to diffuse out of the digestive vacuole. These simulations have been remarkably successful in interpreting most, but not all, of the available experimental data. Their most important success has been in ruling out some interpretations and suggesting experiments to test others. The simulations now need to be extended to describe other antimalarials, to take into account the changing rates of haemoglobin digestion and to provide a more realistic description of the binding.
There is no current work in my laboratory on either ion transport mechanisms in model systems or on the statistical analysis of patch clamp records. However, I still welcome enquiries about these topics.