2000 Global Researcher Conference Proceeding

March 10 - 12, 2000

Conference: 2000 Global Researcher Conference
Title: Consequences of tetramerization and expression levels of Aquaporin-2 in phenotype-genotype correlation studies in autosomal NDI
Authors: van Os, Carel; Kamsteeg, Erik-Jan; Deen, Peter M.T.
Institution: University of Nijmegen
Van Os

Congenital Nephrogenic Diabetes Insipidus, NDI, is caused by mutations in the V2-receptor and in the AQP2-gene. Recently, several NDI families with an autosomal dominant form of inheritance have been reported. Since mutations in autosomal dominant NDI occurred in the C-terminal tail of the AQP2 molecule, we postulated that mutant AQP2 proteins are disturbed in their routing and, by means of forming tetrameric structures with wild-type AQP2, also influence correct routing to the plasma membrane of the heterotetramer. To validate this hypothesis we studied tetramerization of wild type AQP2 and of the mutant AQP2-E258K which has been described in autosomal NDI. As a control we used AQP2-R187C which occurs in a patient with autosomal recessive NDI. AQP2-E258K was shown to be retained in the Golgi complex while AQP2-R187C was retarded in the endoplasmatic reticulum. Sucrose gradient centrifugation of rat and human kidney membrane proteins and subsequent immunoblotting revealed that AQP2 forms tetramers like AQP1. When expressed in oocytes, wild-type AQP2 and AQP2-E258K also form homotetramers, whereas AQP2-R187C can only be detected in a monomeric state. Upon Co-expression, AQP2-E258K, but not AQP2-R187C, was able to form heterotetramers with wild-type AQP2 which than results in disturbed routing of the heterotetramer. These events explain the dominant character of the E258K mutation and the phenotype of autosomal dominant NDI.

Phenotype-genotype correlation studies in oocytes revealed in dominant but not in recessive NDI a specific dominant-negative effect on wild-type (wt) AQP2 water permeability (Pf), but only at low expression levels. We studied the cell-biological basis for this observation. Injection of different amounts of wt-AQP2 cRNAs revealed that a correlation between AQP2 protein levels and Pf is only obtained with low expression levels. In co-expression studies of wt and mutant AQP2 proteins, higher expression levels of AQP2-R187C also exerted a dominant negative effect on the Pf of wt-AQP2. Immunoblot and immuno-precipitation analysis revealed that this dominant negative effect was caused by competitive inhibition of wt-AQP2 expression and by escape of AQP2-R187C from the endoplasmic reticulum, resulting in oligomerization with wt-AQP2. Since many disease-related mutants of multimeric renal membrane transporters and channels are likely to be identified, our data provide important information for studying the effects of such mutants on the activity of wild-type transporters and channels in oocytes.

Normally, CNDI caused by a mutated AQP2 gene is inherited in a recessive manner. That is, the child must receive a mutated AQP2 from each biological parent to inherit the disease. Sometimes, NDI is inherited in a dominant manner, which means the child only has to inherit a single mutated AQP2 gene. van Os, etc., noted that AQP2 mutations in dominant NDI are located in the section of the AQP2 called the C-terminal tail. They hypothesized this would result in two behaviors:

  1. AQP2s carrying a mutation in the C-terminal tail would not be able to travel from the interior of the cell (where they are synthesized) to the cell membrane. (It is by traveling to and inserting themselves within the cell membrane that AQP2s increase the amount of water that can flow through the membrane.)
  2. By binding with normal AQP2s, these mutant AQP2s are able to disturb the normal AQP2s' ability to travel to the cell membrane.

The researchers determined that the normal AQP2 is a combination of four parts. When expressed in laboratory cell cultures, normal AQP2s form an AQP2 out of four like parts. This is called a homotetramer. A mutated AQP2 gene inherited in a dominant fashion, AQP2-E258K, also forms homotetramers. The researchers found the E258K AQP2s don't make it to the cell membrane. Instead, they are retained in a part of the cell interior called the Golgi complex. When the researchers coexpressed normal AQP2s and E258K AQP2s in the same culture, they found that the two different types of AQP2 still combined to form single units of four parts, but this time the parts from both types of AQP2 could combine. This resulted in an AQP2 of four parts, with some of the parts being mutant AQP2s, and some being normal AQP2s. When a molecule is formed from four non-identical parts, it is called a heterotetramer.

When E258K forms heterotetramers with normal AQP2s, the resultant AQP2s are no longer able to travel to the cell membrane. The researchers coexpressed normal AQP2s with a mutant AQP2 that is inherited in a recessive fashion. This mutant could not form heterotetramers with normal AQP2s. The fact that E258K can form hetotetramers with normal AQP2s and thus prevent them from reaching the cell membrane explains why it can cause NDI even though the child bears only one mutant AQP2 gene inherited from one of his or her parents.

van Os, et al., also noted that the ability of the dominant AQP2 mutation to disrupt AQP2 transport to the cell membrane (and thus reduce its ability to let water flow through the cell membrane) occurs when there are low numbers of both in the cell culture. When recessive mutant AQP2s are coexpressed with normal AQP2s in larger amounts, they too can combine with normal AQP2s to disrupt normal function.