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This Article Full Text Full Text (PDF) All Versions of this Article: 582/1/73    most recent jphysiol.2007.128843v1 Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Subramanian, V. S. Articles by Said, H. M. Search for Related Content PubMed PubMed Citation Articles by Subramanian, V. S. Articles by Said, H. M. Related Collections Cellular

¹ Departments of Medicine (Nephrology) and Physiology/Biophysics, University of California, Irvine, CA 92697, USA
² Department of Pharmacology, University of Minnesota Medical School, Minneapolis, MN 55455, USA
³ Department of Veterans Affairs Medical Center, Long Beach, CA 90822, USA

Retinal abnormality and visual disturbances occur in thiamine-responsive megaloblastic anaemia (TRMA), an autosomal recessive disorder caused by mutations in the human thiamine transporter-1 (hTHTR-1). Human retinal pigment epithelial cells play a pivotal role in supplying thiamine to the highly metabolically active retina but nothing is known about the mechanism, regulation or biological processes involved in thiamine transport in these cells. To address these issues, we used human-derived retinal pigment epithelial ARPE-19 cells to characterize the thiamine uptake process. Thiamine uptake is energy- and temperature-dependent, pH-sensitive, Na⁺-independent, saturable at both the nanomolar (apparent K_m, 30 ± 5 nM) and the micromolar (apparent K_m, 1.72 ± 0.3 µM) concentration ranges, specific for thiamine and sensitive to sulfhydryl group inhibition. The diuretic amiloride caused a concentration-dependent inhibition in thiamine uptake, whereas the anti-trypanosomal drug, melarsoprol, failed to affect the uptake process. Both hTHTR-1 and hTHTR-2 are expressed in ARPE-19 cells as well as in native human retinal tissue with expression of the former being significantly higher than that of the latter. Uptake of thiamine was adaptively regulated by extracellular substrate level via transcriptionally mediated mechanisms that involve both hTHTR-1 and hTHTR-2; it was also regulated by an intracellular Ca²⁺–calmodulin-mediated pathway. Confocal imaging of living ARPE-19 cells expressing TRMA-associated hTHTR-1 mutants (D93H, S143F and G172D) showed various expression phenotypes. These results demonstrate for the first time the existence of a specialized and regulated uptake process for thiamine in a cellular model of human retinal pigment epithelia that involves hTHTR-1 and hTHTR-2. Further, clinically relevant mutations in hTHTR-1 lead to impaired cell surface expression or function of the transporter in retinal epithelial ARPE-19 cells.

(Received 22 January 2007; accepted after revision 23 April 2007; first published online 26 April 2007)
Corresponding author H. M. Said: Department of Veterans Affairs, Medical Center-151, Long Beach, CA 90822, USA. Email: hmsaid{at}uci.edu

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