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Effect of ABCC4 Genetic Variation on MRP4 ATPase Activity

  • Author(s): Friedman, Jessica Lauren
  • Advisor(s): Kroetz, Deanna L
  • et al.
Abstract

ATP-binding cassette (ABC) transporters are a diverse family of transmembrane proteins that facilitate the ATP powered translocation of substrates across cellular membranes. Multidrug resistance-associated protein 4 (MRP4) is a highly polymorphic, human ABC transporter with a wide array of substrates, including, antiviral drugs and prostaglandins. Previously, altered antiviral drug transport capacities of nonsynonymous single nucleotide polymorphic MRP4 variants, relative to reference MRP4 transport activity, were reported.

This project focused on characterizing the ATP hydrolysis capabilities of the G487E MRP4 variant (rs11568668); G487 is located in an ATP binding cassette, also known as the nucleotide binding domain, of MRP4. Previously published works report a 0.008 minor allele frequency in Asian American individuals (n = 120) and show decreased transport activity with G487E. Novel data on G487E MRP4 activity from this study corroborate the decreased drug transport findings.

This study characterized prostaglandin E2 – stimulated ATP hydrolysis by human MRP4 reference and G487E variant expressed in insect cells. MRP4 specific activity was determined by its selective inhibition with beryllium fluoride. Reference MRP4 reached a maximal ATP hydrolysis velocity of 27.4 +/- 1.07 nmoles Pi/mg protein/minute; for comparison, ATP hydrolysis in Sf21 membranes absent of exogenous transporters reached a maximum velocity of 18.4 +/- 0.55 nmoles Pi/mg protein/minute. The G487E MRP4 variant had an intermediate Vmax value of 24.4 +/- 0.93 nmoles Pi/mg protein/minute. As measured by an ANOVA, Vmax values for the Sf21, MRP4 reference and G487E MRP4 membranes were significantly different (p < 0.0001).

In contrast, Km values were similar for the three sample types and ranged from 0.34 +/- 0.16 µM for reference MRP4 to 0.44 +/- 0.18 µM for G487E MRP4. Structural rationales for these data were explored using novel homology models of the first nucleotide binding domain of MRP4. The G487E mutation was determined to only affect the MRP4 binding affinity of ATP; prostaglandin E2 binding was not impacted. These results suggest that the reduced transport phenotype of G487E MRP4 relative to reference MRP4 is due, at least in part, to impaired ATP hydrolysis.

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