Misfolding of Mutant Aquaporin-2 Water Channels in Nephrogenic Diabetes Insipidus

Title: Misfolding of Mutant Aquaporin-2 Water Channels in Nephrogenic Diabetes Insipidus
Authors: Tamarappoo, B.K.; Yang, Baoxue; Verkman, Alan S.
Publisher: Journal of Biological Chemistry
Date Published: December 03, 1999
Reference Number: 491
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.
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This translation by the NDI Foundation is to assist the lay reader. To provide a clear, accessible interpretation of the original article, we eliminated or simplified some technical detail and complicated scientific language. We concentrated our translation on those aspects of the article dealing directly with NDI. The NDI Foundation thanks the researchers for their work toward understanding and more effectively treating this disorder.
© Copyright NDI Foundation 2007 (JC)

Aquaporin-2s (AQP2s) act as channels through which water passes. They are located inside the principal cells of the kidney collecting duct. To fulfill their function as water channels, they must travel from inside the cell to the cell's apical membrane. Once at the apical membrane, they are inserted into it, and there the AQP2's allow water to pass through the principal cells into the inner tissue of the kidney. Mutations in the AQP2 gene result in mutant AQP2s unable to perform their function. This is the molecular basis for one of the forms of congenital nephrogenic diabetes insipidus (NDI).

Mutations in AQP2 genes produce AQP2s whose structure differs from that of normal AQP2s. The structural differences (which vary according to differing mutations in the AQP2 genes - to date, more than 20 different mutations of the AQP2 gene have been recorded) produce AQP2s that differ in their functional capacities. For example, some AQP2 gene mutations produce AQP2s that are capable of channeling water, but are incapable of traveling to the apical membrane. Others are unable to act as water channels.

Working with laboratory cell cultures, Tamarappoo, et al., had previously discovered that two NDI-causing AQP2 mutants, T126M and A147T, were capable of letting water flow through them, but they were retained in the endoplasmic reticulum (ER), a kind of quality control center in the principal cell. As they were retained in the ER these AQP2 mutants were degraded (i.e. broken down) more quickly than normal AQP2s. The researchers speculated that since these mutants could channel water, they might be able to do so at their normal job site - the apical cell membrane. They treated the mutants by soaking the cell culture in which they were in with the chemical chaperones glycerol and trimethylamine-N-oxide (TMAO). This enabled the mutant AQP2s to travel to the apical membrane, thus allowing the cells in the culture to be water permeable. In other words, treatment of the mutants with chemical chaperones allowed them to function as normal AQP2s would.

In their current research, the authors tested the hypothesis that NDI-causing AQP2 mutants are misfolded (i.e. they do not have the shape of a normal AQP2) and thus retained in the ER. They further hypothesized that the chemical chaperones are associated with the refolding of the mutant AQP2s into a more normal AQP2 form.

To determine the AQP2s' (both normal and mutant) shapes at the ER, the researchers measured how soluble normal and mutant AQP2s were to detergents. (Detergent insolubility is a function of defective folding and aggregation of misfolded protein units.) They found that the AQP2 mutants, T126M and A147T, were astonishingly resistant to being dissolved by detergent whereas normal AQP2s were 95% dissolved by the same amount of detergent. This suggests that the two mutant AQP2s were folded differently than normal AQP2s. Both these mutants are retained in the ER. Mutant E258K escapes the ER, but is retained in another part of the cell called the Golgi. E258K was just as susceptible to detergent as the normal AQP2.

The researchers also tested the AQP2s to find how sensitive they were to protease, an element that also breaks down AQP2s. The mutants and the normal AQP2s were equally susceptible to protease digestion. This suggests that the misfolding of the AQP2 mutants retained in the ER is not severe enough to expose parts of it that remain unexposed in normal AQP2s.

When the researchers measured the extent to which both normal and mutant AQP2s were degraded in the laboratory cell cultures, they found that AQP2 mutants were degraded significantly more rapidly than normal AQP2s.This suggests that mutant AQP2s are degraded in the ER and normal AQP2s are degraded outside the ER.

The researchers tested the T126M AQP2 while it was in the ER and they found it could act as a water channel. Thus, its misfolding prevented it from leaving the ER, but not from channeling water. In the final step of the research, cells containing the A147T and T126M mutants were incubated in the chemical chaperone, glycerol. This caused the mutants to be able to properly travel to the apical membrane and there function as water channels. These results provide evidence that NDI-causing mutant AQP2 proteins are misfolded, but functional, and that chemical chaperones both correct the mutants folding defects and their ability to reach the apical membrane. They suggest that strategies to facilitate proper protein folding might have a therapeutic effect in treating NDI.