Molecular cloning of canalicular multispecific organic anion
transporter defective in eisai hyperbilirubinemic rats.
Ito, Kousei, Hiroshi Suzuki, Tomoko Hirohashi, Kazuhiko Kume, Takao
Shimizu, and Yuichi Sugiyama.
Faculty of Pharmaceutical Sciences, and b)Faculty of Medicine, The
University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan
APStracts 3:0152G, 1996.
Several organic anions are excreted into the bile via a canalicular
multispecific organic anion transporter (cMOAT), which is
hereditarily defective in mutant rats such as the Eisai
hyperbilirubinemic rat (EHBR) and TR- rat. In the present study, we
cloned cMOAT from the Sprague Dawley (SD) rat liver cDNA library
based on the homology with human multidrug resistance associated
protein (hMRP). cMOAT was encoded by 4623 bp cDNA with a homology of
53.0 % and 46.3 % with hMRP at the cDNA and deduced amino acid level,
respectively. The deduced amino acid sequence was the same as that
cloned in Wistar rats (Paulusma et al. Science 271: 1126, 1996)
except for four amino acid substitutions. By screening the library,
three kinds of cDNA species for cMOAT with the same open reading
frame and different 3'-untranslated region lengths (0.2, 1.5, and 3.5
kbp) were isolated. The Northern blot analysis of poly(A)+ RNA from
the liver revealed that the expression of plural bands (approximately
5, 6 and 8 kb) was defective in EHBR and this may be due to the
presence of these cDNA species. Expression of cMOAT was observed
almost exclusively in the liver and to a lesser extent in the
duodenum, kidney and jejunum. RT-PCR and subsequent sequence analysis
of EHBR liver, kidney, duodenum and jejunum revealed that one base
pair replacement from G to A at nucleotide 2564 resulted in the
introduction of the premature stop codon in all tissues examined.
This mutation was different from that observed in TR- (Paulusma et
al. Science 271: 1126, 1996). Since EHBR and TR- are allelic mutants
and both strains exhibit an autosomal recessive inheritance in the
biliary excretion of organic anions, it was concluded that the
impaired expression of this particular protein is related to the
pathogenesis of hyperbilirubinemia in the mutant animals.
Received 18 June 1996; accepted in final form 26 July 1996.
APS Manuscript Number G247-6.
Article publication pending Am. J. Physiol. (Gastrointest. Liver
Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 21 August 1996