Hong M, Li S, Zhou F, Thomas PE, You G
Putative Transmembrane Domain 12 of the Human Organic Anion Transporter hOAT1 Determines Transporter Stability and Maturation Efficiency. [JOURNAL ARTICLE]
J Pharmacol Exp Ther 2009 Nov 5.
Human organic anion transporter hOAT1 plays a critical role in the body disposition of clinically important drugs. In transmembrane segment (TM) 12, residues Tyr-490, and dileucine Leu-503/Leu-504 were identified to be critical for hOAT1 function. Substitution of Tyr-490 with alanine let to a dramatic reduction in protein expression of hOAT1 and its transport activity. The contribution of the side chain of Tyr-490 to transport activity was then evaluated by replacing this residue with Trp or Phe. Substitution of Tyr-490 with Trp or Phe partially or fully recovered the protein expression of hOAT1 and its transport activity lost by substitution of Tyr-490 with alanine respectively, suggesting that the aromatic ring and the size of the side chain of Tyr-490 are critical for hOAT1 expression and function. Studies with protease inhibitors and pulse-chase labeling further showed that the loss of expression of hOAT1 and its transport activity by replacing Tyr-490 with alanine resulted from accelerated degradation of the transporter, whereas its maturation efficiency was not affected. In contrast to Tyr-490, substitution of Leu-503/Leu-504 with alanine also resulted in complete loss of protein expression of hOAT1 and its transport activity. However, such loss of protein expression could not be prevented by treating mutant-expressing cells with protease inhibitors. Pulse-chase experiments showed that the mutant transporter (L503/L504A) was trapped in the endoplasmic reticulum without conversion into mature form of the transporter. Our results are the first to highlight the central role of TM 12 in maintaining the stability and in promoting maturation efficiency of hOAT1.
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