The Na(+)/K(+)-ATPase1 alpha subunit 3 (ATP1α(3)) is one of many essential components that maintain the sodium and potassium gradients across the plasma membrane in animal cells. Mutations in the ATP1A3 gene cause rapid-onset of dystonia parkinsonism (RDP), a rare movement disorder characterized by sudden onset of dystonic spasms and slowness of movement. To achieve a better understanding of the pathophysiology of the disease, we used immunohistochemical approaches to describe the regional and cellular distribution of ATP1α(3) in the adult mouse brain. Our results show that localization of ATP1α(3) is restricted to neurons, and it is expressed mostly in projections (fibers and punctuates), but cell body expression is also observed. We found high expression of ATP1α(3) in GABAergic neurons in all nuclei of the basal ganglia (striatum, globus pallidus, subthalamic nucleus, and substantia nigra), which is a key circuitry in the fine movement control. Several thalamic nuclei structures harboring connections to and from the cortex expressed high levels of the ATP1α(3) isoform. Other structures with high expression of ATP1α(3) included cerebellum, red nucleus, and several areas of the pons (reticulotegmental nucleus of pons). We also found high expression of ATP1α(3) in projections and cell bodies in hippocampus; most of these ATP1α(3)-positive cell bodies showed colocalization to GABAergic neurons. ATP1α(3) expression was not significant in the dopaminergic cells of substantia nigra. In conclusion, and based on our data, ATP1α(3) is widely expressed in neuronal populations but mainly in GABAergic neurons in areas and nuclei related to movement control, in agreement with RDP symptoms.

Hsp90 is a ubiquitous, ATP-dependent chaperone, essential for eukaryotes. It possesses a broad spectrum of substrates, among which is the p53 transcription factor, encoded by a tumor-suppressor gene. Here, we elucidate the role of the adenine nucleotide in the Hsp90 chaperone cycle, by taking advantage of a unique in vitro assay measuring Hsp90-dependent p53 binding to the promoter sequence. E42A and D88N Hsp90␤ variants bind but do not hydrolyze ATP, whereas E42A has increased and D88N decreased ATP affinity, compared with WT Hsp90␤. Nevertheless, both of these mutants interact with WT p53 with a similar affinity. Surprisingly, in the case of WT, but also E42A Hsp90␤, the presence of ATP stimulates dissociation of Hsp90-p53 complexes and results in p53 binding to the promoter sequence. D88N Hsp90␤ is not efficient in both of these reactions. Using a trap version of the chaperonin GroEL, which irreversibly captures unfolded proteins, we show that Hsp90 chaperone action on WT p53 results in a partial unfolding of the substrate. The ATP-dependent dissociation of p53-Hsp90 complex allows further folding of p53 protein to an active conformation, able to bind to the promoter sequence. Furthermore, in support of these results, the overproduction of WT or E42A Hsp90␤ stimulates transcription from the WAF1 gene promoter in H1299 cells. Altogether, our research indicates that ATP binding to Hsp90␤ is a sufficient step for effective WT p53 client protein chaperoning.Hsp90 is an abundant protein in cells of all known organisms, with the exception of the Archea kingdom. Although in bacteria its presence is not required for survival (1), yeast and higher eukaryotes are fully dependent on its activity (2, 3). In multicellular organisms Hsp90 plays a key role in the activation and stabilization of various protein substrates. Among these are kinases (Raf1, Akt, and Src), telomerase, Rab GDP dissociation inhibitors, glucocorticoid hormone receptors (GR), 3 and transcription factors such as the p53 tumor suppressor protein (4 -6). These Hsp90 substrates belong to different protein families and do not share common sequence or structural motifs. Hence, modes of interaction and chaperoning may possess both common features and specific differences.Hsp90 is functional as a dimer, each monomer consisting of three domains connected with flexible linkers of different length and sequence depending on organism and isoform. The main substrate binding region is proposed to be localized in the middle domain (7), however structural (8) and biochemical studies (9 -11) suggest that at least two distinct surfaces of interaction should exist on Hsp90 chaperone while binding its protein substrate. Repositioning of the domains of the Hsp90 may be translated into conformational rearrangements of a protein substrate, changing its tertiary structure and exposing buried residues, thus enabling interaction with other proteins and ligands, such as hormones or nucleic acids.Despite the initial controversy on the ATP dependence of Hsp90 (12), it was unambig...