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Functional Analysis of Novel Polymorphisms in the Human SLCO1A2 Gene that Encodes the Transporter OATP1A2

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Abstract

The solute carrier organic anion transporting polypeptide 1A2 (OATP1A2, SLCO1A2) is implicated in the cellular influx of a number of drugs. We identified five novel single nucleotide polymorphisms (SNPs) in coding exons of the SLCO1A2 gene in a cohort of subjects: G550A, G553A, G673A, A775C, and G862A, that encoded the OATP1A2 variants E184K, D185N, V255I, T259P, and D288N, respectively. The function and expression of these variant transporters were assessed in HEK-293 cells. We found that the novel variants, E184K, D185N, T259P, and D288N, were associated with impaired estrone-3-sulfate, imatinib, and methotrexate transport (∼20–50% of wild-type control); function was retained by OATP1A2-V255I. From biotinylation assays, the decreased function of these variants was due, at least in part, to impaired plasma membrane expression. The four loss-of-function variants were studied further using mutagenesis to produce variants that encode residues with different charges or steric properties. From immunoblotting, the replacement of negatively charged residues at amino acid positions 184 and 185 impaired membrane expression, while either a positive or negative charge at residue 288 supported the correct membrane targeting of OATP1A2. Replacement of T259 with bulky residues disrupted transporter stability. From molecular models, E184, D185, and D288 were located near several charged residues such that intramolecular ionic interactions may stabilize the transporter structure. Individuals who carry these novel SNPs in the SLCO1A2 gene may be at risk from impaired efficacy or enhanced toxicity during treatment with drugs that are substrates for OATP1A2.

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Abbreviations

OATP1A2:

Organic anion transporting polypeptide 1A2

E3S:

Estrone-3-sulfate

GlpT:

Glycerol 3-phosphate transporter

PBS:

Phosphate-buffered saline

SLCO:

Solute carrier organic anion transporter

SNP:

Single nucleotide polymorphism

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ACKNOWLEDGMENTS

Special thanks to Avy Sim for the assistance with the computer modeling of OATP1A2 structure. This study was supported by grants from Cancer Council NSW and the Australian National Health and Medical Research Council. The generous gifts of imatinib and 14C-imatinib from Novartis are gratefully acknowledged.

Conflict of Interest

The authors declare no conflict of interest.

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Authors and Affiliations

  1. Faculty of Pharmacy, The University of Sydney, Sydney, NSW, 2006, Australia

    Fanfan Zhou, Jian Zheng, Andreas Jodal, Pei H. Cui, W. Bret Church & Michael Murray

  2. Alkali Soil Natural Environmental Science Center, Northeast Forestry University/Key Laboratory of Saline-alkali Vegetation Ecology Restoration in Oil Field, Ministry of Education, Harbin, 150040, China

    Jian Zheng

  3. Retinal Therapeutics Research Group, Save Sight Institute, The University of Sydney, Sydney, NSW, 2000, Australia

    Ling Zhu

  4. Discipline of Pharmacology, School of Medical Sciences, The University of Sydney, Sydney, NSW, 2006, Australia

    Pei H. Cui & Michael Murray

  5. Department of Medical Oncology, Westmead Hospital, Sydney, NSW, 2145, Australia

    Mark Wong & Howard Gurney

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  1. Fanfan Zhou
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Correspondence to Fanfan Zhou.

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Zhou, F., Zheng, J., Zhu, L. et al. Functional Analysis of Novel Polymorphisms in the Human SLCO1A2 Gene that Encodes the Transporter OATP1A2. AAPS J 15, 1099–1108 (2013). https://doi.org/10.1208/s12248-013-9515-1

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  • DOI: https://doi.org/10.1208/s12248-013-9515-1

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