Human organic anion transporter hOAT1 plays a critical role in the body disposition of clinically important drugs.To investigate the role of transmembrane segment (TM) 12 in the function of hOAT 1,we replaced each residue within TM 12 with alanine and analyzed the transport activity in mutant-transfected cells.We discovered several critical residues:Tyr-490,and dileucine Leu-503/Leu-504.Substitution of Tyr-490 with alanine let to a dramatic reduced 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 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,but not its H group are critical for hOAT1 function.Studies using protease inhibitors further showed that the loss of transport activity by replacing Tyr-490 with alanine resulted from accelerated degradation of the mutant transporter at both the cell surface through lysosomal pathway and in the endoplasmic reticulum through proteasomal pathways.In contrast to Tyr-490,substitution of Leu-503/Leu-504 with alanine also resulted in complete loss of transport activity.However,such loss could not be prevented by treating mutant-expressing cells with either lysosomal or proteasomal inhibitors.Pulse-chase experiment showed that the mutant transporter (L503/L504A) was trapped in the endoplasmic reticulum.Our results are the first to highlight the central role of TM 12 in maintaining the stability and in promoting maturation efficiency of hOAT1.