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1 Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, National Institute of Health Reseach Triangle Park, Research Triangle Park, NC, USA
2 Department of Pharmaceutical Sciences, Medical University of South Carolina, Charleston, SC, USA
* To whom correspondence should be addressed. E-mail: pritcha3{at}niehs.nih.gov.
Although membrane vesicle studies have established the driving forces that mediate renal organic anion secretion and the organic anion transporter, Oat1, has now been cloned in several species, its stoichiometry has remained uncertain. In this study, we have used electrophysiology, kinetic measurements, and static head experiments to determine the coupling ratio for Oat1-mediated organic anion/dicarboxylateexchange. Initial experiments demonstrated that uptake of p-aminohippurate (PAH) by voltage clamped Xenopus oocytes expressing rOat1 led to net entry of positive charge, suggesting that coupling was one-to-one. This conclusion was confirmed by kinetic analysis of PAH and glutarate fluxes in native basolateral membrane vesicles from the rat renal cortex, which showed a Hill coefficient of 1. Likewise, static head experiments on the rat vesicles also showed a 1:1 coupling ratio. To confirm these conclusions in a system expressing a single cloned transporter, MDCK cells were stably transfected with the human exchanger, hOAT1. The hOAT1 expressing cell line showed extensive PAH transport, which was very similar in all respects to transport expressed by hOAT1 in Xenopus oocytes. Its Km for PAH was 8 µM and glutarate effectively trans-stimulated PAH transport. When stoichiometry was assessed using plasma membranes isolated from the hOAT1-expressing cells, both kinetic and static head data indicated that hOAT1 also demonstrated 1:1 coupling between organic anion and dicarboxylate.
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