S.M. Mulders

An aquaporin-2 water channel mutant which causes autosomal dominant nephrogenic diabetes insipidus is retained in the Golgi complex.

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.

The exchange of functional domains among aquaporins with different transport characteristics

Abstract Aquaporins are transmembrane proteins that contain six bilayer-spanning domains, connected by loops A to E. The hourglass model predicts that the conserved loops B and E are essential for the formation of the water pore. To test the importance of loops B and E in the determination of the transport characteristics, we exchanged loops B and/or E between AQP0, AQP2, and AQP3. Detailed functional, immunoblot and immunocytochemical analyses of expression in Xenopus oocytes revealed that six out of the nine chimeric aquaporin proteins were not functional, because of misrouting. AQP0 with loop E of AQP2 was not impaired in its routing and revealed a low water permeability equal to that of wild-type AQP0. AQP2 with loop B of AQP0 was also routed normally and gave a high water permeability, similar to that of wild-type AQP2. AQP0 with loops B and E of AQP2 (AQP0–2BE) did not result in an increase in water permeability and was partly misrouted. However, the plasma membrane expression was high enough to expect an increase in water permeability, as loops B and E of AQP2 confer AQP2’s water permeability to AQP0. Although it is unclear for the dual chimera (AQP0–2BE), the parental water permeabilities obtained in oocytes expressing AQP0 with loop E of AQP2 or AQP2 with loop B of AQP0 indicate that, besides loops B and E, other parts of the AQP protein are important in the determination of the characteristics of the channel.

Physiology and pathophysiology of aquaporins

The biophysical properties of water-filled pores in biological membranes have been studied for decades, and this has cumulated in an accurate description of water channel properties [1]. In spite of all this knowledge, the molecular identification of water channels resulted from a serendipitous discovery. Denker et al. [2] copurified a 28-kD protein together with a 32-kD Rhesus antigen from human red blood cells, and this discovery led to the cloning of the first molecular water channel, CHIP28 [3]. CHIP28 appeared to be a member of the major intrinsic protein (MIP) family of intrinsic membrane proteins, named after the first cloned protein of this family, the major intrinsic protein of lens fibre cells [4]. The molecular fingerprint of MIP family members consists of two repeats, presumably the result of an ancient gene duplication event [5]. Each repeat is characterized by a very conserved region in which an NPA box (asparagine–proline–alanine) is unchanged from bacteria to mammals (Fig.1). Owing to this property, new family members were discovered by homology cloning using reverse transcription–polymerase chain reaction (RT–PCR) and primers corresponding to these conserved sequences [6–12]. It is now clear that genes coding for MIP proteins are ubiquitous in nature. For the functional characterization of water channels, theXenopus oocyte expression system has played a dominant role, merely because the osmotic swelling test of oocytes expressing water channels is of appealing simplicity. For MIP family members that were proven to be water selective, a new more appropriate name was chosen and since then water channels discovered in mammalian tissues have been rebaptized as aquaporins 0 to 5, in the rank order of their discovery [13]. In this review, we will focus on publications that appeared in 1994 and 1995 and we restrict ourselves to mam-malian aquaporins. A number of excellent reviews on water channels have recently been published, reflecting the excitement which surrounds the discovery of aquaporins and their role in water homeostasis of the body [14–17].

Localization of the human gene for aquaporin 3 (AQP3) to chromosome 9, region p21→p12, using fluorescent in situ hybridization

The chromosome location of the gene encoding aquaporin 3 (AQP3), which functions as a channel for water and small polar solutes in the basolateral membrane of the collecting duct of the kidney, was determined. In situ hybridization on metaphase chromosomes allowed the assignment of human AQP3 to chromosome 9p21-->p12.