Klonierung und funktionelle Charakterisierung des pOAT1 in ok-Zellen

Klonierung und funktionelle Charakterisierung des pOAT1 in ok-Zellen

Cloning and functional characterization of the pOAT1 in ok-cells

Mark Florian Sendler

Doctoral thesis

Date of Examination: 2015-11-25

Date of issue: 2015-11-25

Advisor: Hagos, Yohannes Prof. Dr.

Referee: Bohnsack, Markus Prof. Dr.

Referee: Schön, Margarete Prof. Dr.

Persistent Address: http://hdl.handle.net/11858/00-1735-0000-0028-8641-A

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Abstract

English

We created and screened a pig kidney cDNA library for the organic anionen transporter 1 (pOAT1) und could isolate two splice variants (pOAT1: EMBL Acc. AJ308234 and pOAT1A: AJ308235). The functional characterization by expression of pOAT1 in Xenopus laevis oocytes and ok-cells showed an [3H]PAH uptake only for pOAT1. The apparrent Km for [3H]PAH in oocytes is 3,75 ± 1,6 µM and in ok-cells it is 8,54 ± 1,2 µM. This uptake was inhibited by 1mM probenecid and 1mM glutarate. The functional characterization revealed an similar cis inhibition of the pOAT1 mediated uptake for [3H]PAH by pyrazionat, urate, glutarate and probenicid as in pig BLMV. We also could show that the chlorid dependent uptake of [3H]PAH in pig BLMV is mediated by the pOAT1 whereas the chlorid independent urate uptake is mediated by the pOAT3. Testing steroids as substrate for the pOAT1 demonstrated a strong inhibition of the [3H]PAH uptake (Ki for estrone sulfate 113± 4,9µM; Ki for DHEA sulfate 116,5± 3,5µM) whereas [3H]estrone sulfate was not transported by the pOAT1.

Keywords: pOAT1; organic anion transporter; ok cells; slc22 family; pig

Schlagwörter: pOAT1; organische Anionen Transporter; ok-Zellen; slc22 Familie; Schwein