Beneficial Renal and Pancreatic Phenotypes in a Mouse Deficient in FXYD2 Regulatory Subunit of Na,K-ATPase

The fundamental role of Na,K-ATPase in eukaryotic cells calls for complex and efficient regulation of its activity. Besides alterations in gene expression and trafficking, kinetic properties of the pump are modulated by reversible association with single span membrane proteins, the FXYDs. Seven members of the family are expressed in a tissue-specific manner, affecting pump kinetics in all possible permutations. This mini-review focuses on functional properties of FXYD2 studied in transfected cells, and on noteworthy and unexpected phenotypes discovered in a Fxyd2-/- mouse. FXYD2, the gamma subunit, reduces activity of Na,K-ATPase either by decreasing affinity for Na+, or reducing Vmax. FXYD2 mRNA splicing and editing provide another layer for regulation of Na,K-ATPase. In kidney of knockouts, there was elevated activity for Na,K-ATPase and for NCC and NKCC2 apical sodium transporters. That should lead to sodium retention and hypertension, however, the mice were in sodium balance and normotensive. Adult Fxyd2-/- mice also exhibited a mild pancreatic phenotype with enhanced glucose tolerance, elevation of circulating insulin, but no insulin resistance. There was an increase in beta cell proliferation and beta cell mass that correlated with activation of the PI3K-Akt pathway. The Fxyd2-/- mice are thus in a highly desirable state: the animals are resistant to Na+ retention, and showed improved glucose control, i.e. they display favorable metabolic adaptations to protect against development of salt-sensitive hypertension and diabetes. Investigation of the mechanisms of these adaptations in the mouse has the potential to unveil a novel therapeutic FXYD2-dependent strategy

A dystonia-like movement disorder with brain and spinal neuronal defects is caused by mutation of the mouse laminin β1 subunit, Lamb1

A new mutant mouse (lamb1t) exhibits intermittent dystonic hindlimb movements and postures when awake, and hyperextension when asleep. Experiments showed co-contraction of opposing muscle groups, and indicated that symptoms depended on the interaction of brain and spinal cord. SNP mapping and exome sequencing identified the dominant causative mutation in the Lamb1 gene. Laminins are extracellular matrix proteins, widely expressed but also known to be important in synapse structure and plasticity. In accordance, awake recording in the cerebellum detected abnormal output from a circuit of two Lamb1-expressing neurons, Purkinje cells and their deep cerebellar nucleus targets, during abnormal postures. We propose that dystonia-like symptoms result from lapses in descending inhibition, exposing excess activity in intrinsic spinal circuits that coordinate muscles. The mouse is a new model for testing how dysfunction in the CNS causes specific abnormal movements and postures. DOI: http://dx.doi.org/10.7554/eLife.11102.00

Impaired AQP2 trafficking in Fxyd1 knockout mice: A role for FXYD1 in regulated vesicular transport

The final adjustment of urine volume occurs in the inner medullary collecting duct (IMCD), chiefly mediated by the water channel aquaporin 2 (AQP2). With vasopressin stimulation, AQP2 accumulation in the apical plasma membrane of principal cells allows water reabsorption from the lumen. We report that FXYD1 (phospholemman), better known as a regulator of Na,K-ATPase, has a role in AQP2 trafficking. Daytime urine of Fxyd1 knockout mice was more dilute than WT despite similar serum vasopressin, but both genotypes could concentrate urine during water deprivation. FXYD1 was found in IMCD. In WT mice, phosphorylated FXYD1 was detected intracellularly, and vasopressin induced its dephosphorylation. We tested the hypothesis that the dilute urine in knockouts was caused by alteration of AQP2 trafficking. In WT mice at baseline, FXYD1 and AQP2 were not strongly co-localized, but elevation of vasopressin produced translocation of both FXYD1 and AQP2 to the apical plasma membrane. In kidney slices, baseline AQP2 distribution was more scattered in the Fxyd1 knockout than in WT. Apical recruitment of AQP2 occurred in vasopressin-treated Fxyd1 knockout slices, but upon vasopressin washout, there was more rapid reversal of apical AQP2 localization and more heterogeneous cytoplasmic distribution of AQP2. Notably, in sucrose gradients, AQP2 was present in a detergent-resistant membrane domain that had lower sedimentation density in the knockout than in WT, and vasopressin treatment normalized its density. We propose that FXYD1 plays a role in regulating AQP2 retention in apical membrane, and that this involves transfers between raft-like membrane domains in endosomes and plasma membranes

Vasopressin induced trafficking of FXYD1 in wild type IMCD <i>in vivo</i>.

<p>Wild type mice were injected with dDAVP (1 μg/kg) and sacrificed at 0 time (a-c), 4 hours (d-f), and 16 hours (g-i) post-injection. Fixed kidneys were sectioned, and slides were stained with antibodies against AQP2 (a,d,g) and PLM-C1 antibodies against FXYD1 (b,e,h). dDAVP stimulated the trafficking of FXYD1 from an intracellular location towards apical membrane. Co-localization of FXYD1 and AQP2 at apical membrane is seen in yellow after 4 hours (f). This figure is representative of 3 independent experiments. Nuclei were labeled with TO-PRO-3. Bars, 10 μm.</p

The daytime defect in urine concentration in Fxyd1^-/- mice was compensated over 24 h.

<p>The daytime defect in urine concentration in Fxyd1^-/- mice was compensated over 24 h.</p

FXYD1 is implicated in urinary concentration.

<p>Box and whisker plot of the osmolality of daytime (afternoon) samples of male mouse WT and knockout (KO) urine at baseline and after water deprivation, showing a difference in baseline osmolality but the near-normal ability of the KO to concentrate its urine. The asterisks indicate a P value of <0.0001, 2-tailed Student’s <i>t</i>-test. When calculated as average ± SEM the results were as follows. WT control conditions, 2,019 ± 143, n = 20; KO control conditions 1,161 ± 123, n = 21; Student’s <i>t</i>-test, P < 0.0001. WT after 36 hours water deprivation, 4,224 ± 140, n = 8; KO water deprivation, 3,716 ± 383, n = 8; <i>t</i>-test, P = 0.26. Female mice were also tested in control conditions, and the results for baseline osmolality were WT, 2,169+/-92, n = 7; KO, 1,085+/-108, n = 7, <i>t</i>-test P < 0.001.</p

AQP2 abundance was reduced in FXYD1 knockout mice.

<p>(A, B) <i>Fxyd1</i>^-/- mice had a lower abundance of AQP2 in inner medulla. Inner medulla from WT and KO mice was obtained as lysates and tested on blots with specific antibodies [representative blot in (A)]. Both core (c) and glycosylated (g) species of AQP2 were reduced in KO animals. (B) quantification of results of three experiments. (C, D) Blot quantification of the relative changes in inner medullary AQP2 levels after 2 hour treatment of mice with dDAVP, 3 experiments. Stimulation with dDAVP resulted in a similar increase of AQP2 recovered in pelleted crude membranes in both WT (C) and KO (D). Bars are ± S.E.M. and significance was evaluated by Student’s <i>t</i>-test.</p