Elsevier

Neurobiology of Disease

Volume 77, May 2015, Pages 88-93
Neurobiology of Disease

A functional correlate of severity in alternating hemiplegia of childhood,☆☆

https://doi.org/10.1016/j.nbd.2015.02.002Get rights and content

Highlights

  • Na+/K+ATPase forward cycling is reduced in alternating hemiplegia of childhood.

  • The function of Na+/K+ATPase is further diminished by negative dominance.

  • Loss of Na+/K+ATPase proton transport correlates with severe disease phenotype.

Abstract

Objective

Mutations in ATP1A3, the gene that encodes the α3 subunit of the Na+/K+ ATPase, are the primary cause of alternating hemiplegia of childhood (AHC). Correlations between different mutations and AHC severity were recently reported, with E815K identified in severe and D801N and G947R in milder cases. This study aims to explore the molecular pathological mechanisms in AHC and to identify functional correlates for mutations associated with different levels of disease severity.

Methods

Human wild type ATP1A3, and E815K, D801N and G947R mutants were expressed in Xenopus laevis oocytes and Na+/K+ ATPase function measured. Structural homology models of the human α3 subunit containing AHC mutations were created.

Results

The AHC mutations examined all showed similar levels of reduction in forward cycling. Wild type forward cycling was reduced by coexpression with any mutant, indicating dominant negative interactions. Proton transport was measured and found to be selectively impaired only in E815K. Homology modeling showed that D801 and G947 lie within or near known cation binding sites while E815 is more distal. Despite its effect on proton transport, E815K was also distant from the proposed proton transport route.

Interpretation

Loss of forward cycling and dominant negativity are common and likely necessary pathomechanisms for AHC. In addition, loss of proton transport correlated with severity of AHC. D801N and G947R are likely to directly disrupt normal Na+/K+ binding while E815K may disrupt forward cycling and proton transport via allosteric mechanisms yet to be elucidated.

Introduction

Alternating hemiplegia of childhood (AHC) is a neurological disorder characterized by repeated attacks of paralysis on one or both sides of the body beginning before 18 months of age. There is a pressing need for effective AHC treatment. Flunarizine, a non-selective calcium channel blocker, is a widely prescribed drug for AHC. Unfortunately, flunarizine only provides symptomatic relief and its efficacy varies between patients (Mikati et al., 2000, Neville and Ninan, 2007, Sweney et al., 2009). The development of AHC therapeutics has been hindered by its complex clinical presentation. There is a well recognized clinical heterogeneity in AHC, where some patients have longer or more frequent hemiplegic attacks in addition to greater extent of cognitive dysfunction and presence of co-morbidities such as seizure and respiratory complications. In contrast, some patients have relatively mild developmental delay and with few or no co-morbidities (Mikati et al., 2000, Sasaki et al., 2014, Yang et al., 2014).

Sequencing studies identified mutations in the gene, ATP1A3, as a primary cause of AHC (Heinzen et al., 2012, Ishii et al., 2013, Rosewich et al., 2012). Three recurring mutations within the gene account for ~ 60% of all AHC cases, D801N, E815K, and G947R. Furthermore, genotype-phenotype analysis revealed that these mutations correlated with clinical severity. In general, patients with D801N or G947R have better clinical outcomes than patients with E815K (Sasaki et al., 2014, Yang et al., 2014). Because the genetic correlation with disease severity is strong, environmental factors are unlikely to play a major role in determining severity. The molecular and functional mechanisms responsible for this clinical heterogeneity are unknown.

ATP1A3 encodes for the α3 subunit of the Na+/K+ ATPase. The α3 subunit is neuron specific, and is highly expressed in the cortex, hippocampus, basal ganglia and thalamus (McGrail et al., 1991). The α3 subunit has 10 transmembrane α-helices which contain the Na+ and K+ binding sites and the cytoplasmic domains involved in ATP hydrolysis (Bublitz et al., 2010). The majority of AHC mutations identified are located within the transmembrane helices (> 70%) (Heinzen et al., 2012). Na+/K+ ATPase critically regulates the Na+ and K+ electrochemical gradients via forward cycling. Forward cycling describes the process by which Na+/K+ ATPase uses ATP hydrolysis to transport three Na+ out and two K+ into the cell (Post et al., 1972). Recently, the Na+/K+ ATPase has also been shown to conduct protons under physiologically relevant conditions. It is proposed that while Na+ ions are leaving the Na+/K+ ATPase during forward cycling, an aqueous path is exposed, which allows protons to passively enter the cell (Vedovato and Gadsby, 2014). This newly revealed function is well positioned to impact neuronal excitability on account of well documented effects of intracellular protons on ion channels and receptors (Church et al., 1998, Takahashi and Copenhagen, 1996, Tombaugh and Somjen, 1996, Traynelis and Cull-Candy, 1991, Waldmann and Lazdunski, 1998).

The functional impact of ATP1A3 mutations on Na+/K+ ATPase have been examined in model systems. Protein blots showed that mutations do not alter α3 subunit membrane expression while enzymatic assays found significant reductions in ATPase and phosphorylation activities, critical steps for proper forward cycling (Heinzen et al., 2012, Weigand et al., 2014). However, the extent of reduction in ATPase and phosphorylation activity was similar between mutations associated with mild and severe AHC. The binding capacity to ouabain, a Na+/K+ ATPase inhibitor, was also examined. Although D801N showed normal ouabain binding capacity it was absent in G947R and E815K (Weigand et al., 2014) and, importantly, no correlation with the disease severity was observed.

On the strength of genetic findings, this study hypothesized that the biophysical changes caused by individual AHC mutations are responsible for the correlations with AHC severity. Human mutations D801N, G947R and E815K, were expressed in Xenopus laevis oocytes and examined using electrophysiological techniques. The properties examined were forward cycling, dominant negativity and proton transport. Homology models of the human α3 subunit were also created to predict the structural-functional impact of mutations. A better understanding of ATP1A3 mutations implicated in AHC may improve clinical diagnosis and prognosis and also revealing novel therapeutic approaches.

Section snippets

Plasmid preparation

ATP1A3 mutations examined in the human α3 subunit (Heinzen et al., 2012) were: c.2401G > A (D801N), c.2839G > C (G947R) and c.2443G > A (E815K). Xenopus laevis atp1b3 was synthesized by Genscript (Piscataway, NJ). Since Xenopus laevis oocytes have endogenous atp1b3, Xenopus laevis atp1b3 was used to avoid creating additional heterogeneity of assembled Na+/K+ ATPases which would have reduced the power to discriminate between various genotypes. All coding sequences were subcloned into an oocyte high

Loss of forward cycling function with AHC mutations

Forward cycling was examined in oocytes expressing the wild type or AHC mutant constructs. Forward cycling current traces were first inspected visually and a consistent reduction in outward current amplitudes was observed in all mutations examined (Fig. 1A) as compared to wild type. Maximum current response was observed at + 40 mV. In comparison to wild type, forward cycling was reduced by 67, 79 and 69% in D801N, G947R and E815K respectively (Fig. 1B). Despite being associated with more severe

Discussion

This study aims to identify pathological mechanisms of ATP1A3 mutations implicated in AHC and a functional basis for AHC clinical heterogeneity. AHC mutations D801N, G947R and E815K, were characterized in vitro by electrophysiology using human α3 subunits. Our data shows that all AHC mutations cause loss of forward cycling of the Na+/K+ ATPase. We also identified that AHC mutations are dominant negative, a novel pathomechanism in the three recurring AHC mutations. Data from this study strongly

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    Conflict of interest and submission declaration: The authors have nothing to disclose. The manuscript is not under consideration for publication in any other journals.

    ☆☆

    Acknowledgment statement: We would like to acknowledge Prof. David C. Gadsby, Dr. Natascia Vedovato, Dr. Peter A. Paulsen and Hanne Poulsen for their helpful suggestions on experimental protocol.

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