Reversed polarized delivery of an aquaporin-2 mutant causes dominant nephrogenic diabetes insipidus

Vasopressin regulates body water conservation by redistributing aquaporin-2 (AQP2) water channels from intracellular vesicles to the apical surface of renal collecting ducts, resulting in water reabsorption from urine. Mutations in AQP2 cause autosomal nephrogenic diabetes insipidus (NDI), a disease characterized by the inability to concentrate urine. Here, we report a frame-shift mutation in AQP2 causing dominant NDI. This AQP2 mutant is a functional water channel when expressed in Xenopus oocytes. However, expressed in polarized renal cells, it is misrouted to the basolateral instead of apical plasma membrane. Additionally, this mutant forms heterotetramers with wild-type AQP2 and redirects this complex to the basolateral surface. The frame shift induces a change in the COOH terminus of AQP2, creating both a leucine- and a tyrosine-based motif, which cause the reversed sorting of AQP2. Our data reveal a novel cellular phenotype in dominant NDI and show that dominance of basolateral sorting motifs in a mutant subunit can be the molecular basis for disease

1,25-Dihydroxyvitamin D₃-independent stimulatory effect of estrogen on the expression of ECaCl in the kidney

Estrogen deficiency results in a negative Ca2+ balance and bone loss in postmenopausal women. In addition to bone, the intestine and kidney are potential sites for estrogen action and are involved in Ca2+ handling and regulation. The epithelial Ca2+ channel ECaCl (or TRPV5) is the entry channel involved in active Ca2+ transport. Ca2+ entry is followed by cytosolic diffusion, facilitated by calbindin-D28K and/or calbindin-D9k, and active extrusion across the basolateral membrane by the Na+/Ca2+-exchanger (NCX1) and plasma membrane Ca2+-ATPase (PMCA1b). In this transcellular Ca2+ transport, ECaCl probably represents the final regulatory target for hormonal control. The aim of this study was to determine whether 17β-estradiol (17β-E2) is involved in Ca2+ reabsorption via regulation of the expression of ECaCl. The ovariectomized rat model was used to investigate the regulation of ECaCl, at the mRNA and protein levels, by 17β-E2 replacement therapy. Using real-time quantitative PCR and immunohistochemical analyses, this study demonstrated that 17β-E2 treatment at pharmacologic doses increased renal mRNA levels of ECaCl, calbindin-D28K, NCX1, and PMCA1b and increased the protein abundance of ECaCl. Furthermore, the involvement of 1,25-dihydroxyvitamin D3 in the effects of 17β-E2 was examined in 25-hydroxyvitamin D3-1α-hydroxylase-knockout mice. Renal mRNA expression of calbindin-D9K, calbindin-D28K, NCX1, and PMCA1b was not significantly altered after 17β-E2 treatment. In contrast, ECaCl mRNA and protein levels were both significantly upregulated. Moreover, 17β-E2 treatment partially restored serum Ca2+ levels, from 1.63 ± 0.06 to 2.03 ± 0.12 mM. In conclusion, this study suggests that 17β-E2 is positively involved in renal Ca2+ reabsorption via the upregulation of ECaCl, an effect independent of 1,25-dihydroxyvitamin D3.</p

Water channel properties of major intrinsic protein of lens

The functions of major intrinsic protein (MIP) of lens are still unresolved; however the sequence homology with channel-forming integral membrane protein (CHIP) and other Aquaporins suggests that MIP is a water channel. Immunolocalizations confirmed that Xenopus oocytes injected with bovine MIP cRNA express the protein and target it to the plasma membrane. Control oocytes or oocytes expressing MIP or CHIP exhibited small, equivalent membrane currents that could be reversibly increased by osmotic swelling. When compared with water-injected control oocytes, the coefficient of osmotic water permeability (Pf) of MIP oocytes was increased 4-5-fold with a low Arrhenius activation energy, while the Pf of CHIP oocytes increased > 30-fold. To identify structures responsible for these differences in Pf, recombinant MIP proteins were expressed. Analysis of MIP-CHIP chimeric proteins revealed that the 4-kDa cytoplasmic domain of MIP did not behave as a negative regulator. Individual residues in MIP were replaced by residues conserved among the Aquaporins, and introduction of a proline in the 5th transmembrane domain of MIP raised the Pf by 50%. Thus oocytes expressing MIP failed to exhibit ion channel activity and consistently exhibited water transport by a facilitated pathway that was qualitatively similar to the Aquaporins but of lesser magnitude. We conclude that MIP functions as an Aquaporin in lens, but the protein may also have other essential functions

Water channel properties of major intrinsic protein of lens

The functions of major intrinsic protein (MIP) of lens are still unresolved; however the sequence homology with channel-forming integral membrane protein (CHIP) and other Aquaporins suggests that MIP is a water channel. Immunolocalizations confirmed that Xenopus oocytes injected with bovine MIP cRNA express the protein and target it to the plasma membrane. Control oocytes or oocytes expressing MIP or CHIP exhibited small, equivalent membrane currents that could be reversibly increased by osmotic swelling. When compared with water-injected control oocytes, the coefficient of osmotic water permeability (Pf) of MIP oocytes was increased 4-5-fold with a low Arrhenius activation energy, while the Pf of CHIP oocytes increased > 30-fold. To identify structures responsible for these differences in Pf, recombinant MIP proteins were expressed. Analysis of MIP-CHIP chimeric proteins revealed that the 4-kDa cytoplasmic domain of MIP did not behave as a negative regulator. Individual residues in MIP were replaced by residues conserved among the Aquaporins, and introduction of a proline in the 5th transmembrane domain of MIP raised the Pf by 50%. Thus oocytes expressing MIP failed to exhibit ion channel activity and consistently exhibited water transport by a facilitated pathway that was qualitatively similar to the Aquaporins but of lesser magnitude. We conclude that MIP functions as an Aquaporin in lens, but the protein may also have other essential functions