Abstract
Multidrug resistance protein 1 (MRP1/ABCC1) is an ATP-dependent efflux pump that can confer resistance to multiple anticancer drugs and transport conjugated organic anions. Unusually, transport of several MRP1 substrates requires glutathione (GSH). For example, estrone sulfate transport by MRP1 is stimulated by GSH, vincristine is co-transported with GSH, or GSH can be transported alone. In the present study, radioligand binding assays were developed to investigate the mechanistic details of GSH-stimulated transport of estrone sulfate by MRP1. We have established that estrone sulfate binding to MRP1 requires GSH, or its non-reducing analogue S-methyl GSH (S-mGSH), and further that the affinity (Kd) of MRP1 for estrone sulfate is 2.5-fold higher in the presence of S-mGSH than GSH itself. Association kinetics show that GSH binds to MRP1 first, and we propose that GSH binding induces a conformational change, which makes the estrone sulfate binding site accessible. Binding of non-hydrolyzable ATP analogues to MRP1 decreases the affinity for estrone sulfate. However, GSH (or S-mGSH) is still required for estrone sulfate binding, and the affinity for GSH is unchanged. Estrone sulfate affinity remains low following hydrolysis of ATP. The affinity for GSH also appears to decrease in the post-hydrolytic state. Our results indicate ATP binding is sufficient for reconfiguration of the estrone sulfate binding site to lower affinity and argue for the presence of a modulatory GSH binding site not associated with transport of this tripeptide. A model for the mechanism of GSH-stimulated estrone sulfate transport is proposed.
Highlights
Addition to transporting these endogenously formed metabolites, MRP1 has an important role in detoxification and protection of normal tissues from xenobiotics (9 –12)
The specific binding of estrone sulfate to MRP1 could be readily measured in the presence of the non-reducing GSH analogue S-methyl GSH (S-mGSH)
We have used this assay to investigate how binding of these organic anions is affected by binding and hydrolysis of ATP
Summary
Materials—[6,7-3H]Estrone sulfate (57.3 Ci/mmol) and [14,15,19,203H]LTC4 were purchased from PerkinElmer Life Sciences. Membrane proteins (10 g) were incubated with [3H]estrone sulfate (35– 40 nM) in the presence of (a) 3 mM S-mGSH and various concentrations of LTC4 (3 nM to 10 M), E217G (300 nM to 3 mM), or methotrexate (1 M to 3 mM) or (b) various concentrations of GSH or S-mGSH (1 M to 10 mM), before filtering as described above. Saturation Binding Isotherms—To determine parameters of maximal binding capacity (Bmax) and binding dissociation constant (Kd), 10 g of membrane protein was incubated with various concentrations of [3H]estrone sulfate (10 nM to 30 M) This assay was done in the presence of 0.3, 1, 3, or 10 mM S-mGSH or GSH (plus 10 mM DTT) in the presence or absence of 1 mM E217G. Differences were considered statistically significant when p was Ͻ0.05
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
