Functional role of the C terminus of human organic anion transporter hOAT1
Wen Xu1, Kunihiko Tanaka, An-qiang Sun
1Department of Pharmaceutics, Rutgers, the State University of New Jersey, Piscataway, New Jersey 08854, USA.
Abstract:
Human organic anion transporter hOAT1 plays critical roles in the body disposition of environmental toxins and clinically important drugs. In the present study, we examined the role of the C terminus of hOAT1 in its function. Combined approaches of cell surface biotinylation and transport analysis were employed for such purposes. It was found that deletion of the last 15 amino acids (residues 536-550) or the last 30 amino acids (residues 521-550) had no significant effect on transport activity. However, deletion of the entire C terminus (residues 506-550) completely abolished transport activity. Alanine scanning mutagenesis within the region of amino acids 506-520 led to the discovery of two critical amino acids: Glu-506 and Leu-512. Substitution of negatively charged Glu-506 with neutral amino acids alanine or glutamine resulted in complete loss of transport activity. However, such loss of transport activity could be rescued by substitution of Glu-506 with another negatively charged amino acid aspartic acid, suggesting the importance of negative charge at this position for maintaining the correct tertiary structure of the transporter, possibly by forming a salt bridge with a positively charged amino acid. Substitution of Leu-512 with amino acids carrying progressively smaller side chains including isoleucine, valine, and alanine resulted in mutants (L512I, L512V, and L512A) with increasingly impaired transport activity. However, the cell surface expression of these mutants was not affected. Kinetic analysis of mutant L512V revealed that the reduced transport activity of this mutant resulted mainly from a reduced maximum transport velocity Vmax without affecting the binding affinity (1/Km) of the transporter for its substrates, suggesting that the size of the side chain at position 512 critically affects transporter turnover number. Together, our results are the first to highlight the central role of the C terminus of hOAT1 in the function of this transporter.
Insights
The C-terminus of human organic anion transporter 1 (hOAT1) is crucial for its function in transporting toxins and drugs. Specific amino acids, Glu-506 and Leu-512, within this region are vital for hOAT1 transporter activity.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- Human organic anion transporter 1 (hOAT1) is essential for the disposition of environmental toxins and drugs.
- Understanding the structural and functional roles of hOAT1 is critical for drug development and toxicology.
Purpose of the Study:
- To investigate the role of the C-terminal region of hOAT1 in its transport function.
- To identify specific amino acid residues within the C-terminus critical for hOAT1 activity.
Main Methods:
- Cell surface biotinylation assays to assess transporter expression.
- Transport assays to measure substrate uptake.
- Alanine scanning mutagenesis and site-directed mutagenesis to probe amino acid function.
- Kinetic analysis (Vmax, Km) to characterize transporter efficiency.
Main Results:
- Deletion of the entire C-terminus (residues 506-550) abolished hOAT1 transport activity.
- Mutations at Glu-506 and Leu-512 significantly impaired or abolished transport.
- Glu-506 requires a negative charge for function, possibly for maintaining tertiary structure.
- Leu-512's side chain size impacts transporter turnover rate (Vmax) without affecting substrate binding (Km).
- Cell surface expression of mutants remained unaffected, indicating functional rather than localization defects.
Conclusions:
- The C-terminus of hOAT1 is indispensable for its transport function.
- Specific residues, Glu-506 and Leu-512, play critical roles in hOAT1 activity, influencing charge and side chain interactions.
- These findings provide novel insights into the structure-function relationship of hOAT1, crucial for understanding drug and toxin transport.
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