The Impact Of Transmembrane Leucine Heptads On Human Organic Anion Transporting Polypeptide 1B1 Transport Funtion And Oligomerization

Organic anion-transporting polypeptides(OATP)play important roles in the uptake of various endogenous and exogenous compounds.OATP1B1,an important member of OATP family,expressed specifically at the basolateral membrane of human hepatocytes,has a great impact on the absorption,distribution and excretion of many clinically important drugs.Our previous studies revealed that,like many other membrane transporters,OATP1B1 form homo-oligomers.Leucine heptad is a kind of conserved amino acid sequence associated with membrane proteins oligomerization.Analysis of OATP1B1 amino acid sequence showed that this transporter possesses three leucine heptads in its transmembrane domains(TMDs).However,whether these motifs affect oligomerization and funtion of OATP1B1 is still largely unclear.In this thesis,we investigated the impact of oligomerization on OATP1B1 transporter function.We also substituted the leucine,isoleucine and valine residues within the leucine heptad motifs in TMD3,TMD6 and TMD8 with alanine,and analyzed the effects of these mutants on uptake function and protein expression as well as oligomerization status of OATP1B1.The results obtained are as follow:(1)We analyzed uptake function of wild-type OATP1B1 when it was co-expressed with a loss-of-function mutant W258 A,and found out that OATP1B1 may function as oligomers in the high affinity binding site of estrone-3-sulfate,while act as monomers for the low affinity binding component of the substrate;(2)Neither single nor multiple mutants of leucine heptad within TMD6 altered uptake function of OATP1B1,suggesting that this leucine heptad has no functional influence on OATP1B1;(3)Only simultaneous mutation of three(L96A/I103A/L110A)or four(L96A/I103A/L110A/F117A)key residues of the leucine heptad within TMD3 resulted in significant reduction of uptake function(trible-mutant)or protein expression(quadro-mutant).Presumably this leucine heptad maintains OATP1B1 expression and function as a unified structure;(4)As long as the leucine heptad mutants within TMD8 contain L378 A,their transport funciton and protein expression would be dramatically decreased.Further study demonstrated that the effect of this leucine heptad on OATP1B1 expression and function was mainly exerted by the Leu378;(5)Chemical cross-linking showed that both TMD3-3A and TMD8-L378 A affected oligomerization of OATP1B1.Co-expression of TMD3-3A with OATP1B1 significantly reduced OATP1B1 uptake function;while co-expression of L378 A resulted in a slightly increased transport activity.These results suggested that these amino acid residues may play important roles in oligomerization through differetmechanisms.Our results implicated the impact of oligomerization on OATP1B1 function,and identified amino acid residues within leucine heptads that are important for expression,function and oligomerization of OATP1 B.These informaiton would help us better understand the functional regulation mechanisms of OATP1B1.