2002 Global Researcher Conference Proceeding
April 26 - 28, 2002
| Conference: | 2002 Global Researcher Conference |
|---|---|
| Title: | Molecular mechanisms underlying dominant Nephrogenic Diabetes Insipidus caused by mutations in the AQP2 gene |
| Authors: | Deen, Peter M.T.; Kamsteeg, Erik-Jan; Marr, Nannette; Konings, Irene B. M.; Lonergan, Michele; Arthus, Marie-Francoise; Nivet, Hubert; van der Sluijs, Peter; Jeck, Nikola; Seyberth, M.D., Hannsjorg W.; Rosenthal, Walter; van Os, Carel; Oksche, Alexander; Bichet, Daniel G. |
| Institutions: | University of Nijmegen, Institut fr Kardiovaskul re Physiologie, Radboud University Nijmegen Medical Center, Hopital du Sacre-Coeur de Montreal, Centre Hospitalier Univ. de Tours, Utrecht University, Universitatskinderklinik, Philipps University, Charite - Universitatsmedizin Berlin, Institut Fuer Pharmakologie |
Autosomal congenital Nephrogenic Diabetes Insipidus (NDI), a disease characterized by the inability of the kidney to concentrate urine in response to AVP, is caused by mutations in the AQP2 water channel. Recently, we identified two novel mutations in the AQP2 gene of families with a dominant inheritance of NDI, which encoded out of frame C-terminal tail, caused by a guanosine deletion (AQP2-727delG) or an adenosine insertion (AQP2-779InsA). Since AQP2 is expressed as a homotetramer in which every monomer is a water channel, it was speculated that these mutants were also retained inside the cell, but were, in contrast to mutants in recessive NDI, able to form heterotetramers with wt-AQP2. Expression of both mutants in oocytes indeed revealed that, though all were properly-folded functional water channels, AQP2-727delG were retained inside the cell. In addition, balanced co-expression studies in oocytes and polarized MDCK cells revealed that both mutants in dominant NDI formed heterotetramers with wt-AQP2, whereas a mutant in recessive NDI did not. The retention of AQP2-E258K in the Golgi region and its heterotetramerization with wt-AQP2 impaired the further routing of wt-AQP2 to the plasma membrane, resulting in a reduced transmembrane water permeability, thereby providing the molecular basis for dominant NDI in this particular family. AQP2-727delG, which was also retained inside the cell, also appeared to reduce the amount of wt-AQP2 in the plasma membrane. A similar effect was seen in double-transfected MDCK cells. The co-localisation of AQP2-727delG, and consequently wt-AQP2, with LAMP-1, however, indicated that NDI in this particular family was caused by the misrouting of wt-AQP2 to lysosomes instead of the Golgi complex region. In contrast to the other mutants in dominant NDI, AQP2-779InsA was almost completely localised in the plasma membrane of oocytes. Since oocytes are not polarized, in contrast to collecting duct cells, it was hypothesized that this mutant might be mistargeted to the basolateral membrane. Indeed, upon expression in MDCK cells, AQP2-779InsA was localised in intracellular vesicles and redistributed to the basolateral membrane upon treatment with the adenylate cyclase activator, forskolin. Upon co-expression with wt-AQP2, both wt-AQP2 and AQP2-779InsA were routed to the basolateral membrane, clearly indicating that in this family mistargeting of wt-AQP2 to the basolateral membrane explained dominant NDI. Expression analysis of several AQP2-779insA mutants in MDCK cells revealed that this basolateral sorting was independently caused by a dileucine-like and tyrosine-based motif.
In conclusion, our expression studies clearly show that in autosomal dominant NDI all mutations, which are found in the C-terminal tail of AQP2, result in functional properly-folded AQP2 mutants that pass the endoplasmic reticulum, but are targeted and retained in another subcellular location than the apical membrane. Their ability to form heterotetramers with wt-AQP2 and the consequent mistargeting of wt-AQP2 explains the dominant inheritance of NDI.
Deen, et al., identified two new mutations of the Aquaporin-2 (AQP2) gene that result in NDI. The researchers were able to discern how the mutations in AQP2 genes prevented the kidney collecting duct cells from allowing sufficient water to pass through their cell walls. Both mutations, AQP2-799InsA actually produced properly folded AQP2 protein capable of letting water channel through them. However, they were still prevented from passing from the cell interior to the proper section of the cell wall where they must be to act as channels for water passage. Both these mutations were able to bind with normal AQPs. When the binding occurred, it prevented the normal AQP2s from traveling to the proper section of the cell wall, thus further reducing the cells' ability to let water pass through them.
The two mutants didn't reach the cell wall for different reasons. AQP2-727delG was routed to lysosomes, an intracellular structure with a different function than the storage vesicles, where the AQP2 should have been routed. AQP2-779InsA was directed to the wrong section of the cell wall. The ability of these mutant AQPs to bind with normal AQPs, and thus prevent the normal AQPs from reaching their job site, explains why the NDI patient need only carry the mutated gene in a single dose (as opposed to two) to manifest symptoms of NDI. This pattern of inheritance is called dominant.