EGF receptor ͉ c-Met ͉ oncogene dependence ͉ proteomics ͉ stable isotope labeling with amino acids in cell culture

The Na؉ ,K ؉ -ATPase catalyzes the active transport of ions. It has two necessary subunits, ␣ and ␤, but in kidney it is also associated with a 7.4-kDa protein, the ␥ subunit. Stable transfection was used to determine the effect of ␥ on Na,K-ATPase properties. When isolated from either kidney or transfected cells, ␣␤␥ had lower affinities for both Na ؉ and K ؉ than ␣␤. A post-translational modification of ␥ selectively eliminated the effect on Na ؉ affinity, suggesting three configurations (␣␤, ␣␤␥, and ␣␤␥*) conferring different stable properties to Na,K-ATPase. In the nephron, segment-specific differences in Na ؉ affinity have been reported that cannot be explained by the known ␣ and ␤ subunit isoforms of Na,K-ATPase. Immunofluorescence was used to detect ␥ in rat renal cortex. Cortical ascending limb and some cortical collecting tubules lacked ␥, correlating with higher Na ؉ affinities in those segments reported in the literature. Selective expression in different segments of the nephron is consistent with a modulatory role for the ␥ subunit in renal physiology.

Phospholemman (FXYD1) is a homolog of the Na,K-ATPase gamma subunit (FXYD2), a small accessory protein that modulates ATPase activity. Here we show that phospholemman is highly expressed in selected structures in the CNS. It is most abundant in cerebellum, where it was detected in the molecular layer, in Purkinje neurons, and in axons traversing the granule cell layer. Phospholemman was particularly enriched in choroid plexus, the organ that secretes CSF in the ventricles, where it colocalized with Na,K-ATPase in the apical membrane. It was also enriched, with Na,K-ATPase, in certain tanycytes or ependymal cells of the ventricle wall. Two different experimental approaches demonstrated that phospholemman physically associated with the Na,K-ATPase in cerebellum and choroid plexus: the proteins copurified after detergent treatment and co-immunoprecipitated from solubilized crude membranes using either anti-phospholemman or anti-Na,K-ATPase antibodies. Phospholemman antibodies precipitated all three Na,K-ATPase alpha subunit isoforms (alpha1-alpha3) from cerebellum, indicating that the interaction is not specific to a particular alpha isoform and consistent with the presence of phospholemman in both neurons and glia. Antibodies against the C-terminal domain of phospholemman reduced Na,K-ATPase activity in vitro without effect on Na+ affinity. At least two other FXYD family members have been detected in the CNS, suggesting that additional complexity of sodium pump regulation will be found.

Na,K-ATPase is an ion transporter that impacts neural and glial physiology by direct electrogenic activity and the modulation of ion gradients. Its three isoforms in brain have cell-type and development-specific expression patterns. Interestingly, our studies demonstrate that in late gestation, the ␣2 isoform is widely expressed in neurons, unlike in the adult brain, in which ␣2 has been shown to be expressed primarily in astrocytes. This unexpected distribution of ␣2 isoform expression in neurons is interesting in light of our examination of mice lacking the ␣2 isoform which fail to survive after birth. These animals showed no movement; however, defects in gross brain development, muscle contractility, neuromuscular transmission, and lung development were ruled out. Akinesia suggests a primary neuronal defect and electrophysiological recordings in the pre-Bö tzinger complex, the brainstem breathing center, showed reduction of respiratory rhythm activity, with less regular and smaller population bursts. These data demonstrate that the Na,K-ATPase ␣2 isoform could be important in the modulation of neuronal activity in the neonate.

Renal Na(+)-K(+)-ATPase is associated with the gamma-subunit (FXYD2), a single-span membrane protein that modifies ATPase properties. There are two splice variants with different amino termini, gamma(a) and gamma(b). Both were found in the inner stripe of the outer medulla in the thick ascending limb. Coimmunoprecipitation with each other and the alpha-subunit indicated that they were associated in macromolecular complexes. Association was controlled by ligands that affect Na(+)-K(+)-ATPase conformation. In the cortex, the proportion of the gamma(b)-subunit was markedly lower, and the gamma(a)-subunit predominated in isolated proximal tubule cells. By immunofluorescence, the gamma(b)-subunit was detected in the superficial cortex only in the distal convoluted tubule and connecting tubule, which are rich in Na(+)-K(+)-ATPase but comprise a minor fraction of cortex mass. In the outer stripe of the outer medulla and for a short distance in the deep cortex, the thick ascending limb predominantly expressed the gamma(b)-subunit. Because different mechanisms maintain and regulate Na(+) homeostasis in different nephron segments, the splice forms of the gamma-subunit may have evolved to control the renal Na(+) pump through pump properties, gene expression, or both.

The gamma-subunit of the Na-K-ATPase is a single-span membrane protein that alters the kinetic properties of the enzyme. It is expressed in the kidney, but our initial observations indicated that it is not present in all nephron segments (Arystarkhova E, Wetzel RK, Asinovski NK, and Sweadner KJ. J Biol Chem 274: 33183-33185, 1999). Here we used triple-label confocal immunofluorescence microscopy in rat kidney with antibodies to Na-K-ATPase alpha1- and gamma-subunits and nephron segment-specific markers. Na-K-ATPase alpha1-subunit stain was low but unambiguous in proximal segments, moderate in macula densa, connecting tubules, and cortical collecting ducts, high in thick ascending limb and distal convoluted tubules, and nearly undetectable in glomeruli, descending and ascending thin limb, and medullary collecting ducts. The gamma-subunit colocalized at staining levels similar to alpha1-subunit in basolateral membranes in all segments except cortical thick ascending limb and cortical collecting ducts, which had alpha1-subunit but no detectable gamma-subunit stain. Selective gamma-subunit expression may contribute to the variations in Na-K-ATPase properties in different renal segments.

The Na,K-ATPase is a dominant factor in retinal energy metabolism, and unique combinations of isoforms of its alpha and beta subunits are expressed in different cell types and determine its functional properties. We used isoform-specific antibodies and fluorescence confocal microscopy to determine the expression of Na,K-ATPase alpha and beta subunits in the mouse and rat retina. In the adult retina, alpha1 was found in Müller and horizontal cells, alpha2 in some Müller glia, and alpha3 in photoreceptors and all retinal neurons. beta1 was largely restricted to horizontal, amacrine, and ganglion cells; beta2 was largely restricted to photoreceptors, bipolar cells, and Müller glia; and beta3 was largely restricted to photoreceptors. Photoreceptor inner segments have the highest concentration of Na,K-ATPase in adult retinas. Isoform distribution exhibited marked changes during postnatal development. alpha3 and beta2 were in undifferentiated photoreceptor somas at birth but only later were targeted to inner segments and synaptic terminals. beta3, in contrast, was expressed late in photoreceptor differentiation and was immediately targeted to inner segments. A high level of beta1 expression in horizontal cells preceded migration, whereas increases in beta2 expression in bipolar cells occurred very late, coinciding with synaptogenesis in the inner plexiform layer. Most of the spatial specification of Na,K-ATPase isoform expression was completed before eye opening and the onset of electroretinographic responses on postnatal day 13 (P13), but quantitative increase continued until P22 in parallel with synaptogenesis.

The ␥ subunit of the Na,K-ATPase, a 7-kDa single-span membrane protein, is a member of the FXYD gene family. Several FXYD proteins have been shown to bind to Na,K-ATPase and modulate its properties, and each FXYD protein appears to alter enzyme kinetics differently. Different results have sometimes been obtained with different experimental systems, however. To test for effects of ␥ in a native tissue environment, mice lacking a functional ␥ subunit gene (Fxyd2) were generated. These mice were viable and without observable pathology. Prior work in the mouse embryo showed that ␥ is expressed at the blastocyst stage. However, there was no delay in blastocele formation, and the expected Mendelian ratios of offspring were obtained even with Fxyd2 ؊/؊ dams. In adult Fxyd2 ؊/؊ mouse kidney, splice variants of ␥ that have different nephron segment-specific expression patterns were absent. Purified ␥-deficient renal Na,K-ATPase displayed higher apparent affinity for Na ؉ without significant change in apparent affinity for K ؉ . Affinity for ATP, which was expected to be decreased, was instead slightly increased. The results suggest that regulation of Na ؉ sensitivity is a major functional role for this protein, whereas regulation of ATP affinity may be context-specific. Most importantly, this implies that ␥ and other FXYD proteins have their effects by local and not global conformation change. Na,K-ATPase lacking the ␥ subunit had increased thermal lability. Combined with other evidence that ␥ participates in an early step of thermal denaturation, this indicates that FXYD proteins may play an important structural role in the enzyme complex.