1999 European Regional Conference Proceeding

May 12 - 16, 1999

Conference: 1999 European Regional Conference
Title: Determination of the functionality of AQP2 missense mutants in recessive NDI
Authors: Marr, Nannette; Kamsteeg, Erik-Jan; van Os, Carel; Deen, Peter M.T.
Institutions: Institut fr Kardiovaskul re Physiologie, University of Nijmegen
Nannette Marr

Several missense mutations in vasopressin-regulated water channel AQP2 have been identified as the cause of autosomal recessive nephrogenic diabetes insipidus (NDI), a disease in which the kidney is unable to concentrate urine in response to vasopressin. Expression studies in oocytes and mammalian cells have shown that misfolding of these AQP2 mutants and consequent impaired export from the endoplasmic reticulum (ER) is the main cause of NDI. Treatment of cells expressing such mutants (e.g. cystic fibrosis transmembrane conductance regulator (CFTR) mutant F508 with chemical chaperones or modulation of natural chaperones in these cells have shown to improve the folding of the mutant proteins, which then can travel to their final destination. In principle, application of drugs exerting such effects provides a basis for development of medicines that could relieve such type of disease.

A first step in this direction is the determination whether AQP2 mutants are functional water channels. Expression in oocytes has shown that two mutants in recessive NDI (AQP2-T126M; AQP2-A147T) conferred water permeability (Pf). However, no conclusions could be made for other mutants, because a method for proper semi-quantification of AQP2 in highly purified plasma membranes (PM) was lacking. Recently, we developed such a method, which we exploited to determine the functionality of different AQP2 missense mutations in recessive NDI (AQP2-G64R, -N68S, -T126M, -A147T, -R187C, -S216P). Injection of high amounts of cRNA revealed that, besides AQP2-T126M and -A147T, also AQP2-G64R was functional. Whereas semi-quantitative immunoblot analysis showed that AQP2-N68S, -R187C and -S216P were significantly higher expressed than wt-AQP2 and since wt-AQP2 expressing oocytes still had a Pf, it can be concluded that AQP2-N68S, -R187C and -S216P are non-functional. In contrast to several previous reports, our data show that mutations in the B-loop (G64R) do not always result in non-functional aquaporins.

After stable transfection and expression in cell lines, it will be investigated whether chemical chaperons and modulation of natural chaperons can restore a normal routing of the functional missense AQP2 mutants to the PM.

Mutations of the aquaporin-2 (AQP2) gene are the cause of autosomal recessive nephrogenic diabetes insipidus (NDI). The AQP2 proteins derived from the mutated AQP2 genes are not properly folded and thus are retained in a part of the principal cells of the kidney collecting duct called the endoplasmic reticulum (ER). Since these misfolded AQP2s are not able to exit from the ER, they are not able to travel to the principal cells' apical membrane, where they must be if they are to function as water channels.

In other diseases that have misfolded proteins retained in the ER, such as cystic fibrosis, researchers have been able to develop chemical chaperons that improve the folding of the misfolded proteins. This improved folding enables the proteins to leave the ER, travel to their final destination, and perform their function. To know whether this approach would be useful in treating autosomal recessive NDI, researchers must first determine whether or not the different AQP2 mutations would be functional after they got out of the ER and reached the apical membrane. It must be determined if the mutant AQP2s could do their job once they got to their job site.

Marr, et al., developed a method to isolate highly purified plasma membranes which can be used to semi-quantify AQP2s. This allowed them to test if misfolded AQP2 proteins caused by specific AQP2 gene mutations could function as water channels. Of the six AQP2 protein mutants they tested, three were found to be functional and three were not. The researchers also found that a mutation in the B-loop of an AQP2 does not, as previously reported, always produce a non-functional AQP2.

Marr, et al., intend to investigate whether chemical chaperons and modulation of natural chaperons can help misfolded but otherwise functional AQP2s leave the ER and travel to the apical membrane.