An Aquaporin-2 Water Channel Mutant Which Causes Autosomal Dominant Nephrogenic Diabetes Insipidus is Retained in the Golgi Complex

Title: An Aquaporin-2 Water Channel Mutant Which Causes Autosomal Dominant Nephrogenic Diabetes Insipidus is Retained in the Golgi Complex
Authors: Morgan, Kenneth; Deen, Peter M.T.; Kamsteeg, Erik-Jan; Lonergan, Michele; Leijendekker, Richtje; van der Sluijs, Peter; Bichet, Daniel G.; Mulders, Sabine M.; Fujiwara, T. Mary; Arthus, Marie-Francoise; van Os, Carel; Rijss, Johan P.L.
Publisher: Journal of Clinical Investigation
Date Published: July 01, 1998
Reference Number: 191
Mutations in the aquaporin-2 (AQP2) water channel gene cause autosomal recessive nephrogenic diabetes insipidus (NDI). Here we report the first patient with an autosomal dominant form of NDI, which is caused by a G866A transition in the AQP2 gene of one allele, resulting in a E258K substitution in the C-tail of AQP2. To define the molecular cause of NDI in this patient, AQP2-E258K was studied in Xenopus oocytes. In contrast to wild-type AQP2, AQP2-E258K conferred a small increase in water permeability, caused by a reduced expression at the plasma membrane. Coexpression of wild-type AQP2 with AQP2-E258K, but not with an AQP2 mutant in recessive NDI (AQP2-R187C), revealed a dominant-negative effect on the water permeability conferred by wild-type AQP2. The physiologically important phosphorylation of S256 by protein kinase A was not affected by the E258K mutation. Immunoblot and microscopic analyses revealed that AQP2-E258K was, in contrast to AQP2 mutants in recessive NDI, not retarded in the endoplasmic reticulum, but retained in the Golgi compartment. Since AQPs are thought to tetramerize, the retention of AQP2-E258K together with wild-type AQP2 in mixed tetramers in the Golgi compartment is a likely explanation for the dominant inheritance of NDI in this patient.
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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)

Every gene comes in pairs, one inherited from each parent. For example, every person's cells (except for egg or sperm cells) contain two aquaporin-2 genes, which produce the protein aquaporin-2 (AQP2). When a gene is mutated, it can produce defective proteins that could lead to a clinical disorder. In many cases, the presence of a mutated gene is masked because its gene pair is normal and produces normal proteins. When this is the case, the mutated gene is called recessive and has little or no influence on the person carrying it. But if both of the AQP2 gene pair are mutated, then there is no normal AQP2 gene to mask the recessive mutated AQP2 gene. These mutated genes can produce defective AQP2 proteins that may result in congenital nephrogenic diabetes insipidus (NDI).

Sometimes only one of a gene pair need be mutated in order to cause an outward abnormality. When this happens, the mutated gene dominates because its presence masks the presence of the other (normal) gene that makes up the gene pair.

Most cases of NDI caused by mutated AQP2 genes are classified as autosomal recessive. The mutant AQP2 gene must be present in a double dose (one from each parent) for NDI to manifest.

Mulders, et al., report on the first known case of an NDI patient with an autosomal dominant form of NDI. This patient, a 37-year-old female, and her 13-year-old daughter, both had a mutation in only one of their AQP2 gene pairs. This mutation, labeled E258K, led to a substitution of a lysine amino acid for a glutamic acid at position 258 in the AQP2 protein. All reported AQP2 mutations that cause recessive NDI are located between the first and last transmembrane helice. The E258K discovered to cause dominant NDI is located in the carboxy terminus tail. This suggests a relationship between the mutation site and the inheritance pattern.

The researchers studied the structure-function relationship in the AQP2 proteins with the E258K mutation. They wanted to see exactly how the change in the AQP2 shape caused by the E258K mutation affected its ability to function. They expressed the mutated AQP2s in laboratory cell cultures, studying how the mutated AQP2s functioned compared to normal AQP2s as well as two other types of mutant AQP2 proteins.

To picture what a normal AQP2 looks like, imagine a beaded string. (The beads are amino acids.) The major portion of the AQP2 lies in six folded clumps (called transmembrane helices 1-6) within the cell membrane, that thin strip of tissue that encircles the cell, separating its insides from the outside. Part of it snakes outside the cell to form three curves called extracellular loops A, C, and E. Part of it snakes inside the cell to form two intracellular loops, B and D. Both ends of the AQP2 are inside the cell with the intracellular loops. One end is called the amino terminus and the other is called the carboxy terminus. (Please look at a diagram of a normal AQP2.)

Normally, the AQP2 is synthesized within the cell in the endoplasmic reticulum (ER). When signaled by the antidiuretic hormone arginine vasopressin (AVP), the AQP2 proteins are transported from the ER to the Golgi Complex. After that they are transported to vesicles which take them to the cell membranes. The vesicles fuse to the membranes and the AQP2s are then inserted into the cell membranes to make them more permeable to water. This increased water permeability allows the kidney collecting ducts to maintain water balance by reabsorbing water and concentrating urine. This does not happen in a patient with NDI.

The researchers found, as expected, that the mutated AQP2 gene that causes recessive NDI produced AQP2s that, due to their structural abnormalities, were retained in the ER and were not able to travel to their work site on the cell membrane surface. The E258K AQP2s could not reach their work site either, but were retained in the Golgi apparatus instead of the ER.

The researchers speculated that the change from a negative to a positive charge in the E258K mutation caused by the substitution of lysine for glutamic acid did not directly interfere with the AQP2's ability to get to the cell membrane. Rather, it signaled the Golgi apparatus to either retain the mutated AQP2 or direct it back inside the cell.