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

ATP1A3 encodes the alpha 3 isoform of Na,K-ATPase. In the brain, it is expressed only in neurons. Human ATP1A3 muta-tions produce a wide spectrum of phenotypes, but particular syndromes are associated with unique substitutions. For argi-nine 756, at the junction of membrane and cytoplasmic do-mains, 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 wild -type, reduced affinity for Na+, and increased affinity for K+. There was modest endoplasmic reticulum retention during biosynthesis at 37 degrees C but little benefit from the folding drug phenylbutyrate (4-PBA), suggesting a tolerated level of mis-folding. When cells were incubated at just 39 degrees C, however, alpha 3 protein level dropped without loss of beta subunit, paralleled by an increase of endogenous alpha 1. Elevated temperature resulted in internalization of alpha 3 from the surface along with some beta sub-unit, accompanied by cytoplasmic redistribution of a marker of lysosomes and endosomes, lysosomal-associated membrane protein 1. After return to 37 degrees C, alpha 3 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 resis-tance as an anchor, forming hydrogen bonds among four lin-early distant parts of the Na,K-ATPase structure. Taken together, our observations are consistent with fever-induced symptoms in patients.