Resequencing of ABCC4 indicated a high degree of polymorphism. Among nine other ABC transporters that were resequenced in similar populations (ABCC1, ABCC2, ABCC3, ABCC5, ABCC6, ABCB1, ABCB4, ABCB11 and ABCG2), only ABCC6 has more variants. Considering only haplotypes inferred from coding and UTR variants, several blocks of variable sites are evident, in particular within exon 8, and across exons 22 to 23 and 26 to 31. The linkage disequilibrium patterns for the 14 variable sites in these 45 haplotypes is similar across all ethnic groups (data not shown), suggesting that this diversity happened in the ancestral population.
Genetic variation at synonymous sites is high while it is relatively low and similar to other ABC transporters at non-synonymous sites. The low πNS/πS ratio suggests that this gene has a low tolerance for non-synonymous compared to synonymous variations. This could explain why most non-synonymous variants are rare. However, one may infer from this low ratio that a mutation affecting the protein structure would be deleterious, but this has not been confirmed clinically. Similar to most ABC efflux transporters, no disease has been associated with a non-functional MRP4.
This study confirms the role of MRP4 in transporting AZT and PMEA by a direct measurement of transport in cells transfected with a known ABCC4 sequence. The lipophilic ester prodrug of PMEA, bis(POM)-PMEA, has been used to increase passive intracellular uptake (Srinivas et al., 1993
; Hatse et al., 1998
). PMEA is then rapidly released intracellularly from the prodrug by esterases (Srinivas et al., 1993
) and is effluxed unchanged by MRP4 (Schuetz et al., 1999
). Endogenous MRP4 protein expressed in HEK 293T could contribute to the cellular efflux. This contribution seems greater in the case of AZT which shows a less dramatic difference between empty vector and MRP4 transfected cells than PMEA. Other transporters are likely to be involved in AZT and PMEA uptake and efflux (OAT1 and MRP5 for PMEA (Cihlar et al., 1999
; Wijnholds et al., 2000
), OAT1−4 and MXR for AZT (Takeda et al., 2002
; Wang et al., 2004
)) and passive diffusion of these antivirals will be determined by their lipophilicity. Differences in these alternate pathways for AZT and PMEA transport may explain the differences observed between these two substrates. MRP4 expression was also high in other cell lines, including MDCKII, CHO-K1, HepG2, and CV-1 cells. Moreover, because MRP4 is highly expressed in the kidney (van Aubel et al., 2002
), HEK 293T cells provide a realistic model for studying the function of this transporter.
MRP4-mediated transport of AZT and PMEA was observed with concentrations as low as 20 nM. Reported Cmax
values are 3.7 μM following a 200 mg dose of AZT (Singlas et al., 1989
) and 67 nM for a 10 mg dose of PMEA (http://www.hepsera.com
). Therefore, an intermediate donor concentration of 100 nM was used in the first screen of the variants. All ten non-synonymous variants of MRP4 that were tested are functional. Four variants have significantly lower function, with the G187W variant displaying the greatest reduction in function. In contrast, the C956S variant had a significantly higher transport of PMEA. In all cases, a similar trend was observed with both substrates; this strongly suggests that the functional differences observed are real and substrate-independent within this same chemical group. The velocity curves can in theory be used to estimate and compare kinetic parameters (Vmax
) related to the transport of AZT and PMEA by MRP4 variant and reference proteins. However, the assay described in this study is an indirect measurement of the efflux, and is based on a difference in accumulation between transfected and untransfected cells. The sensitivity of the analytical method did not allow direct measurement of the amount of substrate being effluxed following accumulation. Therefore, while the Vmax
values could in theory reflect efflux by MRP4 (because the transport velocities related to MRP4 variants and reference have been normalized to empty vector transfected cells), the Km
values would only be apparent and correspond to the extracellular concentration to which the cells are exposed (instead of the intracellular concentration to which the transporter is really exposed).
A mutant designed to be non-functional by replacing a highly conserved glycine in NBD1 with an aspartate showed no difference in intracellular accumulation compared to the empty vector-transfected cells, confirming that the reduction of substrate accumulation seen with the reference and the variants is due to efflux by MRP4. This validates the MRP4 efflux data for AZT and PMEA and is consistent with earlier results with P-gp and MRP1, where a mutation in a specific conserved amino acid in one of the two NBDs is sufficient to lead to a total loss of function (Bakos et al., 1997
; Ren et al., 2004
). The glycine at the fourth position of the ABC signature is almost fully conserved in the ABC transporter family (it is present in 99% of 1000 transporters analyzed in different species).
The functional differences observed with most of the variants cannot be attributed to differences in expression of the proteins at the membrane or total MRP4 expression levels. One exception to this is the G187W variant, which had lower levels of antiretroviral transport and showed deceased expression by Western blotting. A similar observation has been made for several MRP2, OATP1B1 and OATP1A2 variants (Tirona et al., 2001
; Hirouchi et al., 2004
; Lee et al., 2005
). Interestingly, the non-functional mutant is also correctly expressed at the membrane, showing that its loss of function is not due to a decreased stability or impaired trafficking to the membrane. What are other possible mechanisms to explain the functional differences? One hypothesis implicates the modifications in the chemical structure of the protein when replacing one amino acid with another. The Grantham value for G187W is the greatest among the non-synonymous variants of MRP4 (D=184), indicating that this variant has the greatest structural change, with respect to composition, polarity, and molecular volume (Grantham, 1974
). This could contribute to a certain extent to the reduction in function. It is supported by the fact that the G487E and C956S variants also have high Grantham values (D=98 and 102, respectively) and altered functions. However, this estimator can probably not account completely for these data, since another variant with a high Grantham value (K304N) does not show any functional difference.
The ~ 50% reduction in function observed with the G187W variant could be clinically relevant, while the small differences observed with the other variants (G487E, P78A, P403L and C956S) are less likely to be significant. The G187W variant is present at a frequency of 2.5−13% in various ethnic groups. A decrease in MRP4 transport of antivirals could be beneficial as a result of higher target cell concentrations but could also result in a higher incidence of toxicity related to increased systemic and/or tissue levels of MRP4 substrates. The G187W MRP4 variant could also play a role at the organism level, since MRP4 is expressed in the kidney and the liver where it may be involved in antiviral elimination (Zamek-Gliszczynski et al., 2006
; Imaoka et al., 2007
). The expression of MRP4 variants in the liver has been evaluated in a recent clinical study, which showed no association between MRP4 polymorphism and RNA or protein expression in human liver (Gradhand et al., 2008
). Interestingly, there was a trend toward decreased protein levels of 187W MRP4 in normal livers, consistent with our Western blot data. AZT is primarily eliminated by hepatic metabolism before being excreted in the urine, while PMEA is mainly renally excreted (http://us.gsk.com/products/assets/us_retrovir.pdf
). Therefore, the consequences of altered MRP4 transport on AZT and PMEA pharmacokinetics should be investigated.
Previous reports have shown a certain degree of substrate specificity associated with variants in membrane transporters (Erdman et al., 2006
; Jeong et al., 2007
), including a different specificity between endogenous substrates and drugs (Urban et al., 2006
). This specificity should be further investigated for MRP4 which has both physiological and xenobiotic substrates. Inhibitory interactions of xenobiotics with MRP4 non-synonymous variants should also be considered. This is particularly relevant for nucleoside/nucleotide analogs used for treating HIV infection that are always administered as combination therapy.
In summary, ABCC4 is a highly polymorphic gene, as illustrated by the large number of variants and haplotypes identified in this study. Convincing evidence of AZT and PMEA transport by MRP4 is provided in this first functional characterization of MRP4 non-synonymous variants. None of the ten non-synonymous variants of MRP4 which were studied are completely deleterious. The G187W variant shows the greatest decrease in function and lower expression in this in vitro system. The clinical consequences of this altered function (e.g. increased response or higher incidence of side effects) require further investigation.