Temperature instability of a mutation at a multi-domain junction in Na,K-ATPase isoform ATP1A3 (p.Arg756His) produces a fever-induced neurological syndrome

TY - JOUR

T1 - Temperature instability of a mutation at a multi-domain junction in Na,K-ATPase isoform ATP1A3 (p.Arg756His) produces a fever-induced neurological syndrome

AU - Arystarkhova, Elena

AU - Toustrup-Jensen, Mads S

AU - Holm, Rikke

AU - Ko, Jae-Kyun

AU - Lee, Kyung Eun

AU - Feschenko, Polina

AU - Ozelius, Laurie J

AU - Brashear, Allison

AU - Vilsen, Bente

AU - Sweadner, Kathleen J

N1 - Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.

PY - 2023/1

Y1 - 2023/1

N2 - ATP1A3 encodes the α3 isoform of Na,K-ATPase. In the brain, it is expressed only in neurons. Human ATP1A3 mutations produce a wide spectrum of phenotypes, but particular syndromes are associated with unique substitutions. For arginine 756, at the junction of membrane and cytoplasmic domains, mutations produce encephalopathy during febrile infections. Here we tested the pathogenicity of p.Arg756His (R756H) in isogenic mammalian cells. R756H protein had sufficient transport activity to support cells when endogenous ATP1A1 was inhibited. It had half the turnover rate of wildtype, reduced affinity for Na +, and increased affinity for K +. There was modest endoplasmic reticulum retention during biosynthesis at 37 °C but little benefit from the folding drug phenylbutyrate (4-PBA), suggesting a tolerated level of misfolding. When cells were incubated at just 39 °C, however, α3 protein level dropped without loss of β subunit, paralleled by an increase of endogenous α1. Elevated temperature resulted in internalization of α3 from the surface along with some β subunit, accompanied by cytoplasmic redistribution of a marker of lysosomes and endosomes, lysosomal-associated membrane protein 1. After return to 37 °C, α3 protein levels recovered with cycloheximide-sensitive new protein synthesis. Heating in vitro showed activity loss at a rate 20- to 30-fold faster than wildtype, indicating a temperature-dependent destabilization of protein structure. Arg756 appears to confer thermal resistance as an anchor, forming hydrogen bonds among four linearly distant parts of the Na,K-ATPase structure. Taken together, our observations are consistent with fever-induced symptoms in patients.

AB - ATP1A3 encodes the α3 isoform of Na,K-ATPase. In the brain, it is expressed only in neurons. Human ATP1A3 mutations produce a wide spectrum of phenotypes, but particular syndromes are associated with unique substitutions. For arginine 756, at the junction of membrane and cytoplasmic domains, mutations produce encephalopathy during febrile infections. Here we tested the pathogenicity of p.Arg756His (R756H) in isogenic mammalian cells. R756H protein had sufficient transport activity to support cells when endogenous ATP1A1 was inhibited. It had half the turnover rate of wildtype, reduced affinity for Na +, and increased affinity for K +. There was modest endoplasmic reticulum retention during biosynthesis at 37 °C but little benefit from the folding drug phenylbutyrate (4-PBA), suggesting a tolerated level of misfolding. When cells were incubated at just 39 °C, however, α3 protein level dropped without loss of β subunit, paralleled by an increase of endogenous α1. Elevated temperature resulted in internalization of α3 from the surface along with some β subunit, accompanied by cytoplasmic redistribution of a marker of lysosomes and endosomes, lysosomal-associated membrane protein 1. After return to 37 °C, α3 protein levels recovered with cycloheximide-sensitive new protein synthesis. Heating in vitro showed activity loss at a rate 20- to 30-fold faster than wildtype, indicating a temperature-dependent destabilization of protein structure. Arg756 appears to confer thermal resistance as an anchor, forming hydrogen bonds among four linearly distant parts of the Na,K-ATPase structure. Taken together, our observations are consistent with fever-induced symptoms in patients.

KW - Na,K-ATPase

KW - ataxia

KW - cytopathology

KW - fever

KW - hypotonia

KW - mutation pathogenicity

KW - neurodegeneration

KW - phenotype-genotype relationship

KW - thermal inactivation

KW - Temperature

KW - Protein Isoforms/metabolism

KW - Humans

KW - Animals

KW - Sodium-Potassium-Exchanging ATPase/metabolism

KW - Mammals/metabolism

KW - Brain Diseases

KW - Mutation

U2 - 10.1016/j.jbc.2022.102758

DO - 10.1016/j.jbc.2022.102758

M3 - Journal article

C2 - 36462665

SN - 0021-9258

VL - 299

JO - The Journal of Biological Chemistry

JF - The Journal of Biological Chemistry

IS - 1

M1 - 102758

ER -