| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2006: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 2005: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Research Abstract |
In the epithelial tissues such as small intestine, kidney and liver, various organic anion transporters with distinct characteristics were localized to the brush-border and basolateral membranes, and play an important role in determining the unidirectional transport (absorption or secretion) of drugs. However, the mechanism of plasma membrane targeting of organic anion transporters was not fully understood. In the present study, organic anion transporter OAT-K was selected as a model transporter, and we constructed the expression vector in which enhanced green fluorescent protein (EGFP) was fused to the N-terminus of OAT-K. EGFP-OAT-K fusion protein was stably expressed in MDCK cells and its biosynthesis and cell surface targeting were examined. Our data showed that OAT-K was modified by post-translational processing and its C-terminal portion was targeted to the apical membrane. It was suggested that processing of OAT-K occurred at the endoplasmic reticulum and that its N-terminal portion was degraded by proteasomal pathway.
Other organic anion transporters, OAT1 and OAT3, are abundantly expressed in the human kidney, and therefore we examined the mechanisms concerning their regulation of expression by promoter analysis. It was demonstrated that a cAMP-response element exists in the promoter region of OAT3 gene. Transcription factors, CREB-1 and ATF-1, regulate the constitutive and inducible expression via a cAMP-response element, while OAT1 gene was shown to be regulated by hepatocyte nuclear factor-4α. We also succeeded in cloning of MATE1 and MATE2-K, the H^+/organic cation antiporters expressed in the brush-border membrane, and demonstrated their tissue distribution and functional characteristics.
We will compare the mechanisms of membrane targeting between brush-border type and basolateral type of organic ion transporters in the future.
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