Department of Biomedicine, Aarhus University, DK-8000 Aarhus C, Denmark.
Florey Institute of Neuroscience and Mental Health, Parkville, 3052 Victoria, Australia.
J Biol Chem. 2019 Jan 4;294(1):269-280. doi: 10.1074/jbc.RA118.004591. Epub 2018 Nov 8.
The cerebellar ataxia, areflexia, pes cavus, optic atrophy, and sensorineural hearing loss (CAPOS) syndrome is caused by the single mutation E818K of the α3-isoform of Na,K-ATPase. Here, using biochemical and electrophysiological approaches, we examined the functional characteristics of E818K, as well as of E818Q and E818A mutants. We found that these amino acid substitutions reduce the apparent Na affinity at the cytoplasmic-facing sites of the pump protein and that this effect is more pronounced for the lysine and glutamine substitutions (3-4-fold) than for the alanine substitution. The electrophysiological measurements indicated a more conspicuous, ∼30-fold reduction of apparent Na affinity for the extracellular-facing sites in the CAPOS mutant, which was related to an accelerated transition between the phosphoenzyme intermediates EP and EP. The apparent affinity for K activation of the ATPase activity was unaffected by these substitutions, suggesting that primarily the Na-specific site III is affected. Furthermore, the apparent affinities for ATP and vanadate were WT-like in E818K, indicating a normal E-E equilibrium of the dephosphoenzyme. Proton-leak currents were not increased in E818K. However, the CAPOS mutation caused a weaker voltage dependence of the pumping rate and a stronger inhibition by cytoplasmic K than the WT enzyme, which together with the reduced Na affinity of the cytoplasmic-facing sites precluded proper pump activation under physiological conditions. The functional deficiencies could be traced to the participation of Glu-818 in an intricate hydrogen-bonding/salt-bridge network, connecting it to key residues involved in Na interaction at site III.
小脑性共济失调、无反射、高弓足畸形、视神经萎缩和感觉神经性耳聋（CAPOS）综合征是由α3 同工型 Na,K-ATP 酶的单一突变 E818K 引起的。在这里，我们使用生化和电生理方法研究了 E818K、E818Q 和 E818A 突变体的功能特征。我们发现这些氨基酸取代降低了泵蛋白胞质侧部位的表观 Na 亲和力，而这种效应在赖氨酸和谷氨酰胺取代（3-4 倍）比丙氨酸取代更为明显。电生理测量表明，CAPOS 突变体的胞外侧部位的表观 Na 亲和力显著降低约 30 倍，这与磷酸酶中间体 EP 和 EP 之间的快速转换有关。ATP 酶活性的表观 K 激活亲和力不受这些取代的影响，表明主要是 Na 特异性的 III 部位受到影响。此外，E818K 中的 ATP 和钒酸盐的表观亲和力与 WT 相似，表明去磷酸化酶的 E-E 平衡正常。质子泄漏电流在 E818K 中没有增加。然而，CAPOS 突变导致泵速的电压依赖性减弱和胞质 K 的抑制作用增强，与 WT 酶相比，这与胞质侧部位的 Na 亲和力降低一起，使得在生理条件下无法适当激活泵。功能缺陷可追溯到 Glu-818 参与复杂的氢键/盐桥网络，将其与涉及 III 部位 Na 相互作用的关键残基连接起来。
