A functional correlate of severity in alternating hemiplegia of childhood☆,☆☆
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
References (36)
In and out of the cation pumps: P-type ATPase structure revisited
Curr. Opin. Struct. Biol.
(2010)Thermal denaturation of the Na,K-ATPase provides evidence for alpha-alpha oligomeric interaction and gamma subunit association with the C-terminal domain
J. Biol. Chem.
(2001)- et al.
Comparison of the substrate dependence properties of the rat Na, K-ATPase alpha 1, alpha 2, and alpha 3 isoforms expressed in HeLa cells
J. Biol. Chem.
(1991) Polarity and hydrophobicity interactions in protein synthesis process
J. Theor. Biol.
(2006)Subunit stoichiometry of a mammalian K + channel determined by construction of multimeric cDNAs
Neuron
(1992)Alternating hemiplegia of childhood: clinical manifestations and long-term outcome
Pediatr. Neurol.
(2000)P2X7 purinoceptor expression in Xenopus oocytes is not sufficient to produce a pore-forming P2Z-like phenotype
FEBS Lett.
(1997)Activation by adenosine triphosphate in the phosphorylation kinetics of sodium and potassium ion transport adenosine triphosphatase
J. Biol. Chem.
(1972)Heterozygous de-novo mutations in ATP1A3 in patients with alternating hemiplegia of childhood: a whole-exome sequencing gene-identification study
Lancet Neurol.
(2012)A novel ATP1A3 mutation with unique clinical presentation
J. Neurol. Sci.
(2014)
Modulation of neuronal function by intracellular pH
Neurosci. Res.
H(+)-gated cation channels: neuronal acid sensors in the NaC/DEG family of ion channels
Curr. Opin. Neurobiol.
Alternating Hemiplegia of Childhood mutations have a differential effect on Na(+), K(+)-ATPase activity and ouabain binding
Biochim. Biophys. Acta
The phenotypic spectrum of rapid-onset dystonia-parkinsonism (RDP) and mutations in the ATP1A3 gene
Brain
pH modulation of Ca2 + responses and a Ca2 +-dependent K + channel in cultured rat hippocampal neurones
J. Physiol.
A novel recurrent mutation in ATP1A3 causes CAPOS syndrome
Orphanet J. Rare Dis.
Comparative protein structure modeling using Modeller
Curr Protoc Bioinforma
De novo mutations in ATP1A3 cause alternating hemiplegia of childhood
Nat. Genet.
Cited by (0)
- ☆
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.