Water Channels Encoded by Mutant Aquaporin-2 Genes in Nephrogenic Diabetes Insipidus are Impaired in Their Cellular Routing

Title: Water Channels Encoded by Mutant Aquaporin-2 Genes in Nephrogenic Diabetes Insipidus are Impaired in Their Cellular Routing
Authors: Deen, Peter M.T.; Croes, Huib; van Aubel, Remon A.M.H.; Ginsel, Leo A.; van Os, Carel
Publisher: Journal of Clinical Investigation
Date Published: May 01, 1995
Reference Number: 68
Congenital nephrogenic diabetes insipidus is a recessive hereditary disorder characterized by the inability of the kidney to concentrate urine in response to vasopressin. Recently, we reported mutations in the gene encoding the water channel of the collecting duct, aquaporin-2 (AQP-2) causing an autosomal recessive form of nephrogenic diabetes insipidus (NDI). Expression of these mutant AQP-2 proteins (Gly64Arg, Arg187Cys, Ser216Pro) in Xenopus oocytes revealed nonfunctional water channels. Here we report further studies into the inability of these missense AQP-2 proteins to facilitate water transport in Xenopus oocytes. cRNAs encoding the missense AQPs were translated with equal efficiency as cRNAs encoding wild-type AQP-2 and were equally stable. Arg187Cys AQP2 was more stable and Gly6-4Arg and Ser216Pro AQP2 were less stable when compared to wild-type AQP2 protein. On immunoblots, oocytes expressing missense AQP-2 showed, besides the wild-type 29 kDa band, an endoplasmic reticulum-retarded form of AQP-2 of approximately 32 kD. Immunoblots and immunocytochemistry demonstrated only intense labeling of the plasma membranes of oocytes expressing wild-type AQP-2. Therefore, we conclude that in Xenopus oocytes the inability of Gly64-Arg, Arg187Cys or Ser216Pro substituted AQP-2 proteins to facilitate water transport is caused by an impaired routing to the plasma membrane.
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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)

Nephrogenic diabetes insipidus (NDI) is characterized by the inability of the kidney to concentrate urine in response to the message from the antidiuretic hormone, vasopressin (VP). The most common form of congenital NDI is X-linked NDI. In this form of NDI, mutations of the V2 receptor gene, which is located on the X chromosome, result in defective V2 receptors that cannot respond to VP. Far less common is the non-X-linked form of NDI. Here, mutants of the aquaporin-2 (AQP2) gene, which is located on a non-sex chromosome, result in defective AQP2 unable to transport water across cell membranes in the principal cells of the kidney collecting duct.

In this study, Deen, et al., analyzed the AQP2 genes of four NDI patients in whom mutations of the V2 receptor gene were excluded or unlikely. They found one of the patients had a deletion mutation in his AQP2 gene and three patients had a missense mutation in their AQP2 genes. Missense mutations produce a different amino acid than expected in part of the protein produced by the affected gene. The amino acid substitution generated by this mutation results in a misformed or, in some other way, non-functional AQP2. A deletion mutation occurs when one part of the gene, in this case, one nucleotide, is missing.

When these mutants were expressed in cells in the laboratory, all appeared non-functional in that they did not increase the water permeability of test cell membranes. More specifically, the mutant AQP2 did not increase the water permeability of the cell membranes above that of water-injected control cells, whereas normal AQP2 increased the water permeability of cell membranes at least ten times above the water-injected controls.

The researchers tested to be sure the mutant AQP2's inability to transfer water across cell membranes in their experiment was not due to any factors or conditions of the experiment itself. It was not. At least three missense AQP2 mutations' inability to transport water was because they could not get to their work site at the cell membrane. It is hypothesized that under normal conditions AQP2s are located inside the cell near the membrane. They have to be shuttled to their work site by a complex trafficking mechanism. If the AQP2s are not properly shaped, the endoplasmic reticulum (ER), the cellular structure in which the AQP2 is synthesized, folded and modified, will not let them out, and they cannot get to the cell membrane to make it more water permeable. Since they cannot get to their work site, it cannot as yet be determined if they would work if they could just get there. In other words, there is a possibility that these mutations of the AQP2s might still leave them capable of serving as water transporting proteins that would increase the cell membrane's water permeability. But this possibility cannot, as yet, be tested. Further research is required to understand more clearly the molecular and cellular defects in NDI caused by mutations in the AQP2 gene.