FIGURE 8.

ITM2B mutants associated with familial Danish dementia (FDD), retinal dystrophy (FRD) or N-glycosylation (ITM2B-N170Q) exhibit attenuated inhibition of urate uptake by GLUT9 isoforms. (A) Western blot analyses of lysates of oocytes expressing GLUT9a/b or co-expressing GLUT9a/b and ITM2B-myc or myc-tagged mutants of ITM2B associated with familial British dementia (FBD), FDD, FRD or N-glycosylation (N170Q) in Xenopus oocytes. GAPDH protein band acts as a loading control. (B) The [¹⁴C]-urate uptake activity of GLUT9 isoforms expressed alone or co-expressed with ITM2B or its mutants associated with FBD, FDD, FRD or N-glycosylation (N170Q), was measured in ND96 medium. ^∗P < 0.001 compared with urate uptake in presence of normal ITM2B. (C) The [¹⁴C]-urate transport activity of GLUT9 isoforms expressed alone or co-expressed with ITM2B or mutants of ITM2B, was measured in membrane-depolarized oocytes (in Na⁺-free medium containing 98 μM KCl). Note that ITM2B showed proportionate inhibition in both membrane-polarized and depolarized oocytes (Na⁺-free medium containing 98 μM KCl). ^∗P < 0.001 compared with urate uptake mediated by GLUT9 in presence of normal ITM2B. (D) ITMB stimulates urate efflux mediated by GLUT9 isoforms but the ITM2B(N170Q) does not. For [¹⁴C]-urate efflux experiment in Xenopus laevis oocytes, each oocyte was pre-injected with 50 nl of 1500 μM [¹⁴C]-urate and then subjected to urate efflux for 1h in ND96 medium. ^∗P < 0.001 compared with urate efflux mediated by GLUT9 in absence of ITM2B. NS, statistically non-significant. (E) Co-immunoprecipitation of GLUT9a/b with myc-tagged ITM2B or its mutants, from lysates of oocytes co-expressing GLUT9a/b and ITM2B-myc or its mutants. Each oocyte was microinjected with 50 nl of cRNA solution containing 12.5 ng of GLUT9a/b cRNA or a mixture of cRNAs containing 12.5 ng of GLUT9a/b cRNA and 12.5 ng of cRNA of ITM2B or its mutants. (F) Co-immunoprecipitation of GLUT9a with ITM2B-myc or ITM2B(N170Q)-myc, from lysates of oocytes co-expressing GLUT9a and ITM2B-myc or ITM2B(N170Q)-myc. GLUT9 isoforms were immunodetected by Western blotting using rabbit anti-GLUT9 antibody and ITM2B-myc or myc-tagged ITM2B mutant was immunodetected using rabbit anti-Myc antibody. IB, immunoblotting; IP, immunoprecipitation.