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

Missense mutations in ATP1A3, the alpha 3 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 alpha 1 and inducible exogenous alpha 3 were constructed to compare mutation properties. Na,K-ATPase is made in the endoplasmic reticulum (ER), but the glycan-free catalytic a subunit complexes with glycosylated beta 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 beta 1 subunit, impaired trafficking of alpha 3, 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 alpha 3 (ERAD); larger differences in Na,K-ATPase subunit distributions among subcellular fractions; and greater inactivation of eIF2 alpha, a major defensive step of the unfolded protein response. In L924P there was also altered subcellular distribution of endogenous a1 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 alpha 3 expression, and restored morphology.