Misfolding, altered membrane distributions, and the unfolded protein response contribute to pathogenicity differences in Na,K-ATPase ATP1A3 mutations

Elena Arystarkhova,Laurie J Ozelius,Allison Brashear,Kathleen J Sweadner

doi:10.1074/jbc.ra120.015271

Abstract

Missense mutations in ATP1A3, the α3 isoform of Na,K-ATPase, cause neurological phenotypes that differ greatly in symptoms and severity. A mechanistic basis for differences is lacking, but reduction of activity alone cannot explain them. Isogenic cell lines with endogenous α1 and inducible exogenous α3 were constructed to compare mutation properties. Na,K-ATPase is made in the endoplasmic reticulum (ER), but the glycan-free catalytic α subunit complexes with glycosylated β subunit in the ER to proceed through Golgi and post-Golgi trafficking. We previously observed classic evidence of protein misfolding in mutations with severe phenotypes: differences in ER retention of endogenous β1 subunit, impaired trafficking of α3, and cytopathology, suggesting that they misfold during biosynthesis. Here we tested two mutations associated with different phenotypes: D923N, which has a median age of onset of hypotonia or dystonia at 3 years, and L924P, with severe infantile epilepsy and profound impairment. Misfolding during biosynthesis in the ER activates the unfolded protein response, a multiarmed program that enhances protein folding capacity, and if that fails, triggers apoptosis. L924P showed more nascent protein retention in ER than D923N; more ER-associated degradation of α3 (ERAD); larger differences in Na,K-ATPase subunit distributions among subcellular fractions; and greater inactivation of eIF2α, a major defensive step of the unfolded protein response. In L924P there was also altered subcellular distribution of endogenous α1 subunit, analogous to a dominant negative effect. Both mutations showed pro-apoptotic sensitization by reduced phosphorylation of BAD. Encouragingly, however, 4-phenylbutyrate, a pharmacological corrector, reduced L924P ER retention, increased α3 expression, and restored morphology.

Highlights

Heterozygous mutations in the genes for three Na,KATPase catalytic subunit isoforms can result in debilitating neurologic syndromes, but the presentations vary widely in severity
Isogenic cells expressing exogenous α3WT, D923N, and L924P were grown with constant tetracycline induction for at least five passages, and lysates were resolved on Western blots for detection with antiα3 and anti-β1 antibodies (Fig. 1A)
If reduction or inactivation of pump activity were the only consequence of ATP1A3 mutation it would be difficult to understand why some mutations do not manifest until adulthood, whereas others produce symptoms at birth

Summary

Introduction

Heterozygous mutations in the genes for three Na,KATPase catalytic subunit isoforms can result in debilitating neurologic syndromes, but the presentations vary widely in severity. Eleven different mutations were tested initially, and notably, the severity of the human phenotype did not correlate with whether there was enough residual α3 activity at the membrane to keep the cells alive after inhibition of endogenous α1 with ouabain [15].

Results

Conclusion