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Coupling of organic anion transport to the glutamate gradient by OATs and OATPs

Reference	BB/L020823/1
Principal Investigator / Supervisor	Professor Rohan Lewis
Co-Investigators / Co-Supervisors	Dr Ita O'Kelly, Dr Bram Gijsbert Sengers
Institution	University of Southampton
Department	Human Development and Health
Funding type	Research
Value (£)	350,289
Status	Completed
Type	Research Grant
Start date	27/07/2014
End date	26/07/2017
Duration	36 months

Abstract

The organic anion transporter (OAT) and transporting peptide (OATP) families mediate transport of organic anions across cell membranes by exchange. OATs and OATPs transport endogenous substrates (e.g. hormones, metabolites) and drugs (e.g. statins, anti-cancer drugs) in cells throughout the body. In order to accumulate a substrate within a cell, via exchange, its transport must be coupled to an opposing gradient of second substrate. The gradients which drive OAT/OATP transporters are not clearly defined. We have demonstrated that human OAT4 and OATP2B1 expressed in Xenopus oocytes mediate glutamate efflux. Glutamate concentrations within cells are 50-100 x those in plasma. We hypothesise that uptake of OAT4 and OATP2B1 substrates are coupled to glutamate efflux and that the glutamate gradient may drive other members of the OAT and OATP families. This project will use Xenopus oocytes to determine which other human members of the OAT/OATP families mediate glutamate efflux and hence characterise transporter activity and kinetics of relevant transporters. The oocyte studies will determine whether these transporters meditate glutamate efflux but not how they function in response the conditions within human cells. Subcellular compartmentalisation or intracellular affinity may limit glutamate's ability to drive OAT activity. For this reason our second aim will be to determine what the exchange substrates are in cells over-expressing OAT/OATP transporters. Finally, we will use exchanger models developed as part of our current BBSRC funded study to model OAT4 and OATP2B1 activity in cells and transporting epithelia. This multidisciplinary project addresses the essential, but often overlooked, role of gradients in driving secondary active uptake by exchangers. It will deliver key information on the gradients driving OAT/OATP activity allowing physiologically based computational modelling of these transporters applicable to cells throughout the body.

Summary

The organic anion transporter (OAT) and functionally related organic anion transporting peptide (OATP) families transport a wide range of biologically and pharmacologically important molecules into and out of cells. They transport hormones, metabolites, waste products and many drugs including aspirin, antibiotics, anticancer, cholesterol and blood pressure lowering drugs. They mediate the intestinal absorption of drugs into the body, the transport these drugs into target cells and finally the removal of these drugs from the body via the kidneys and liver. For example, the reason that some medications cannot be taken with grapefruit juice is that it inhibits specific OATPs. This project will investigate how these transporters obtain the energy they require from chemical gradients to undertake their essential biological and pharmacological roles. OAT and OATPs can transport substances both into and out of cells. If you want to pump a drug into a cell, transporting in both directions is inefficient (as what comes in can go straight out again). However, by coupling transporter activity to a second substrate it is possible to mediate transfer of the drug in one direction (as the substance of interest is taken up but cannot get out again because the second substrate is using the transporter). For this to work the second substrate must have a higher concentration within the cell than the substance in question. Glutamate concentrations within the cell are 50-100 x higher than in plasma and we have found that OAT4 and OATP2B1 transport glutamate. Thus the glutamate gradient could drive uptake of drugs and other molecules transported by OAT4 and OATP2B1. In an analogous manner to the release of water stored behind a dam, when the glutamate is released from the cell the potential energy stored in the gradient can be captured and used to drive the uptake of drugs. There are multiple OAT and OATP transporters many of which transport the same molecules. If some of these transporters are coupled to the glutamate gradient, and some are not, then even though they transport the same substrates they will act in different ways and play different roles in the body. Those being coupled to the glutamate gradient (e.g. OAT4) would mediate uptake of drugs and hormones while those which are not would be more likely to mediate efflux. The first aim of this project will be to determine which other members of the OAT and OATP families transport glutamate. The fact that OAT4, OATP2B1 or any other OAT/OATP, transports glutamate does not necessarily mean that its activity will be coupled to the glutamate gradient; this will depend on the kinetics of glutamate transfer, the presence of other substrates and the subcellular localisation of glutamate. The second aim of this project will be to determine when a specific substrate is taken up, then what intracellular molecules is it exchanged for in human cells. It can become difficult to predict how a transporter will function if its activity depends on the concentrations of multiple different substrates on two sides of a biological membrane. It is even more complicated when there are different transporters for the same substrates in the same membrane or in apical and basal membranes of an epithelial barrier. The third aim of this project is to develop a physiologically based mathematical model of how these transporters work. This will allow us to predict how they will function together and how this will affect the transport of their endogenous substrates and of therapeutic agents. The OAT and OATP families play essential biological roles and are important for our understanding of normal cellular function and drug pharmacology. This project explores the extent to which glutamate, which has a large trans-membrane gradient compared to other OAT/OATP substrates, drives the activity of these transporters and provide mathematical models which will help us explain and predict their function throughout the body.

Impact Summary

This project will generate new knowledge about the biological and pharmacological roles of organic anion transporters. It will advance our knowledge about how the body handles drugs and toxins and how transporters work as part of biological systems. This knowledge will benefit academics, pharmaceutical companies and regulatory agencies and ultimately the consumers through improved safety and efficacy of the drugs they are prescribed. Academic impact: The academic impact of this work will come from the intellectual advances made by the work, the skills developed by staff and applicants and the contribution to the UK's developing excellence in the area of transporter modelling. The training of highly skilled researchers and the experience it will provide for systems biologists and engineers to work in an interdisciplinary team. The innovative models developed by this project will be readily applicable to the study of transport in all living systems. Through interactions with other researchers in the UK this project will enhance the UK's excellence in transporter biology. This impact of the work will be enhanced by application of innovative approaches to mathematical modelling of complex systems which will be made available to researchers through the European Bioinformatics Institute (EBI) Biomodels Database. These findings will be communicated nationally and internationally through presentation at conferences and publication in high impact peer reviewed journals. This work will make a significant contribution to worldwide academic advancement and the UK's capacity to undertake research in these and related areas. Health Impacts: The health impacts of this work will come from improved understanding of the underlying physiology of disease and the pharmacology drug action. This understanding will be translated into improved drug treatment regimes designed to have greater efficacy and fewer side effects. This project addresses the cross council research priority area of lifelong health and wellbeing. One of the main impacts of this work will be to inform the development of physiologically based pharmacokinetic models of drug action. These models help to predict the effects and side effects of these drugs and to understand how drug-drug interactions may affect their efficacy or side effect profiles. As such the work will be of use to researchers, drug companies and regulatory agencies including the Medicines and Healthcare Products Regulatory Agency (MHRA) in the UK and Food and Drug Administration (FDA) in the USA. In conjunction with personal genetic profiling, this would allow the development of optimised personalised treatment regimes. These findings will also be relevant to veterinary medicine. Commercial impact: The commercial impact of this work will come through utilisation of the knowledge gained regarding the function of these transporters and its application to pharmacokinetics, drug discovery and personalised medicine. This will be communicated to the commercial sector through attendance at conferences with significant industry participation (e.g. the Biomedical Transporter Conference), through University of Southampton Business Fellows and through publications in academic Journals. The models will be available to industry through the EBI Biomodels Database. This work has particular relevance to the pharmaceutical industry as the OATs and OATPs transport clinically important therapeutic agents. Increasing our knowledge of how drugs are taken up, transported and eliminated form the body along with models of these processes may aid the development of new drugs and allow better strategies for the use of current drugs including the development of personalised medicine regimes. Summary: The academic, heath and commercial impacts of this work will combine to have economic and societal impacts. Improved heath benefits the individual's quality of life and their ability to make social and economic contributions to society.

Committee	Research Committee A (Animal disease, health and welfare)
Research Topics	Systems Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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