ATP1A3-Disease Mutations at the Ion Binding Sites Unravel Sequential Release of Na+ in the HNA+/K+ ATPase Alpha 3

Abstract

The Na+/K+ATPase uses the energy of ATP hydrolysis to export 3Na+ from the cell and import 2K+. During the transport cycle the ions are temporarily occluded within the protein before being release to the opposite site. Crystal structure analysis have identified three ion binding sites (in which the ions are occluded) within the transmembrane region of the protein named I, II and III. Sites I and II are almost identical for Na+ and two K+ and site site III is exclusive for Na+. Mutations in the ATP1A3 gene, are associated to several neurological disorders of increasing severity including cerebellar ataxia, areflexia, optic atrophy, and sensorineural hearing loss (CAPOS), fever-induced paroxysmal weakness and encephalopathy (FIPWE), rapid dystonia parkinsonism (RDP) and alternating hemiplegia of childhood (AHC). The location of the mutation seems to have a crucial role on the disease severity as mutations targeting residues contributing to the coordination of the ions or near the ion binding sites including many AHC mutations tend to associate with a more severe phenotype compared to the cytoplasmic mutations linked to the CAPOS and FIPWE syndromes. Previously we showed that by studying Na+-mediated pre-steady state kinetics of the human Na+/K+ATPase alpha 3 currents we could identify three components of relaxation (fast, medium and slow) representing the sequential binding/release and occlusion/deocclusion of each Na+. Here, we use known and newly identified ATP1A3 mutations directly involved or near the proposed Na+ binding sites to determine whether we can assign relaxation components with specific binding sites. At the same time, by characterizing many ATP1A3 we expect to identify key features in the mutant pumps that could help us to better understand the molecular mechanism behind the phenotypical variability observed in patients.