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. The R756H protein had sufficient transport activity to support cells when endogenous ATP1A1 was inhibited. It had approximately 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^oC, suggesting a tolerated level of misfolding, and little benefit from the folding drug phenylbutyrate (4-PBA). When cells were incubated in the febrile range at 39^oC, however, α3 protein level dropped significantly 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, LAMP1. After return to 37^oC, α3 protein levels recovered with cycloheximide-sensitive new protein synthesis. Heating in vitro showed activity loss at a rate 20-30-fold faster than the wild type, indicating a temperature-dependent destabilization of protein structure. Arg756 appears to confer thermal resistance by 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, followed by recovery with some persistent symptoms.

ATP1A3编码 Na,K-ATPase 的 α3 亚型。在大脑中，它仅在神经元中表达。人类ATP1A3突变产生广泛的表型，但特定的综合征与独特的替代相关。对于精氨酸 756，在膜和细胞质结构域的连接处，突变会在发热性感染期间产生脑病。在这里，我们测试了 p.Arg756His (R756H) 在等基因哺乳动物细胞中的致病性。当内源性 ATP1A1 受到抑制时，R756H 蛋白具有足够的转运活性来支持细胞。它的周转率大约是野生型的一半，对 Na ^{+ 的}亲和力降低，对 K ^{+ 的}亲和力增加。^{在 37 o}的生物合成过程中存在适度的内质网保留C，表明可容忍的错误折叠水平，折叠药物苯丁酸 (4-PBA) 几乎没有益处。^{然而，当细胞在 39 o} C的发热范围内孵育时，α3 蛋白水平显着下降而没有 β 亚基的损失，同时内源性 α1 的增加。升高的温度导致 α3 与一些 β 亚基从表面内化，伴随着溶酶体和核内体标记物 LAMP1 的细胞质重新分布。回到 37 ^o C 后，α3 蛋白水平随着环己酰亚胺敏感的新蛋白合成而恢复。体外加热显示活性损失的速度比野生型快 20-30 倍，表明蛋白质结构的温度依赖性不稳定。Arg756 似乎通过在 Na,K-ATPase 结构的四个线性远距离部分之间形成氢键来赋予耐热性。综上所述，我们的观察结果与患者发烧引起的症状一致，随后恢复并伴有一些持续性症状。