Misfolding of mutant aquaporin-2 water channels in nephrogenic diabetes insipidus

We reported that several aquaporin-2 (AQP2) point mutants that cause nephro genic diabetes insipidus (NDI) are retained in the endoplasmic reticulum (E R) of transfected mammalian cells and degraded but can be rescued by chemic al chaperones to function as plasma membrane water channels (Tamarappoo, B. K., and Verkman, A. S. (1998) J. Clin. Invest. 101, 2257-2267). To test wh ether mutant AQP2 proteins are misfolded, AQP2 folding was assessed by comp arative detergent extractability and limited proteolysis, and AQP2 degradat ion kinetics was measured by label-pulse-chase and immunoprecipitation, In ER membranes from transfected CHO cells containing [S-35]methionine-labeled AQP2, mutants T126M and A147T were remarkably detergent-resistant; for exa mple wild-type AQP2 was >95% solubilized by 0.5% CHAPS whereas T126M was <1 0% solubilized, E258K, an NDI-causing AQP2 mutant which is retained in the Gels, is highly detergent soluble like wild-type AQP2. The mutants and wild -type AQP2 were equally susceptible to digestion by trypsin, thermolysin, a nd proteinase K. Stopped-flow light scattering measurements indicated that T126M AQP2 at the ER was fully functional as a water channel. Pulse-chase s tudies indicated that the increased degradation rates for T126M (t(1/2) 2.5 h) and A147T (2 h) compared with wildtype AQP2 (4 h) involve a brefeldin A -resistant, ER-dependent degradation mechanism. After growth of cells for 4 8 h in the chemical chaperone glycerol, AQP2 mutants T126M and A147T became properly targeted and relatively detergent-soluble. These results provide evidence that NDI-causing mutant AQP2 proteins are mis folded, but function al, and that chemical chaperones both correct the trafficking and folding d efects. Strategies to facilitate protein folding might thus have therapeuti c efficacy in NDI.