The putative transmembrane segment 7 of human organic anion transporter hOAT1 dictates transporter substrate binding and stability.

Human organic anion transporter hOAT1 plays a critical role in the body disposition of clinically important drugs. We examined the role of the putative transmembrane segment (TM) 7 in the function of hOAT1. Each residue within putative TM7 was replaced by alanine, and the uptake of para-aminohippurate was studied in cells expressing the mutants. We discovered four critical amino acid residues: Trp-346, Thr-349, Tyr-353, and Tyr-354. Substitution of Tyr-353 and Tyr-354 with alanine led to the loss of transport activity without affecting the surface expression of the transporter, whereas substitution of Trp-346 and Thr-349 with alanine lead to the loss of the total expression of the transporter. The effect of side chains of Tyr-353 and Tyr-354 on transporter functions were further evaluated by replacing these residues with Phe or Trp. Among all the mutants studied (Y353W, Y353F, Y354W, and Y354F), only mutant Y353F regained 30% transport activity, which was lost from replacement of Tyr-353 with alanine, suggesting that both the -OH group and the size of the side chain at positions 353 and 354 are critical for maintaining the full transport activity. To investigate the mechanisms underlying the loss of total protein expression when Trp-346 and Thr-349 were replaced with alanine, mutant-expressing cells were treated with lysosomal or proteasomal inhibitors. Our results showed that only proteasomal inhibitors resulted in the accumulation of mutant proteins, indicating that proteasome is involved in the degradation of the mutant transporters. Therefore, Trp-346 and Thr-349 are critically involved in the stability of the transporter.