We reported that several aquaporin-2 (AQP2) point mutants that cause nephrogenic diabetes insipidus (NDI) are retained in the endoplasmic reticulum (ER) of transfected mammalian cells and degraded but can be rescued by chemical chaperones to function as plasma membrane water channels (Tamarappoo, B. K., and Verkman, A. S. (1998) J. Clin. Invest. 101, 2257-2267). To test whether mutant AQP2 proteins are misfolded, AQP2 folding was assessed by comparative detergent extractability and limited proteolysis, and AQP2 degradation kinetics was measured by label-pulse-chase and immunoprecipitation. In ER membranes from transfected CHO cells containing [(35)S]methionine-labeled AQP2, mutants T126M and A147T were remarkably detergent-resistant; for example wild-type AQP2 was >95% solubilized by 0.5% CHAPS whereas T126M was <10% solubilized. E258K, an NDI-causing AQP2 mutant which is retained in the Golgi, is highly detergent soluble like wild-type AQP2. The mutants and wild-type AQP2 were equally susceptible to digestion by trypsin, thermolysin, and proteinase K. Stopped-flow light scattering measurements indicated that T126M AQP2 at the ER was fully functional as a water channel. Pulse-chase studies indicated that the increased degradation rates for T126M (t((1)/(2)) 2.5 h) and A147T (2 h) compared with wild-type AQP2 (4 h) involve a brefeldin A-resistant, ER-dependent degradation mechanism. After growth of cells for 48 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 misfolded, but functional, and that chemical chaperones both correct the trafficking and folding defects. Strategies to facilitate protein folding might thus have therapeutic efficacy in NDI.