Junghans Cornelia, Vukojević Vladana, Tavraz Neslihan N, Maksimov Eugene G, Zuschratter Werner, Schmitt Franz-Josef, Friedrich Thomas
Technical University of Berlin, Institute of Chemistry, Berlin, Germany.
Department of Clinical Neuroscience, Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden.
Biophys J. 2017 Nov 21;113(10):2249-2260. doi: 10.1016/j.bpj.2017.08.053. Epub 2017 Oct 5.
The Na,K-ATPase is a plasma membrane ion transporter of high physiological importance for ion homeostasis and cellular excitability in electrically active tissues. Mutations in the genes coding for Na,K-ATPase α-subunit isoforms lead to severe human pathologies including Familial Hemiplegic Migraine type 2, Alternating Hemiplegia of Childhood, Rapid-onset Dystonia Parkinsonism, or epilepsy. Many of the reported mutations lead to change- or loss-of-function effects, whereas others do not alter the functional properties, but lead to, e.g., reduced protein stability, reduced protein expression, or defective plasma membrane targeting. Na,K-ATPase frequently assembles with other membrane transporters or cellular matrix proteins in specialized plasma membrane microdomains, but the effects of these interactions on targeting or protein mobility are elusive so far. Mutation of established interaction motifs of the Na,K-ATPase with ankyrin B and caveolin-1 are expected to result in changes in plasma membrane targeting, changes of the localization pattern, and of the diffusion behavior of the enzyme. We studied the consequences of mutations in these binding sites by monitoring diffusion of eGFP-labeled Na,K-ATPase constructs in the plasma membrane of HEK293T cells by fluorescence correlation spectroscopy as well as fluorescence recovery after photobleaching or photoswitching, and observed significant differences compared to the wild-type enzyme, with synergistic effects for combinations of interaction site mutations. These measurements expand the possibilities to study the consequences of Na,K-ATPase mutations and provide information about the interaction of Na,K-ATPase α-isoforms with cellular matrix proteins, the cytoskeleton, or other membrane protein complexes.
钠钾ATP酶是一种质膜离子转运体，对电活性组织中的离子稳态和细胞兴奋性具有高度的生理重要性。编码钠钾ATP酶α亚基同工型的基因突变会导致严重的人类疾病，包括2型家族性偏瘫性偏头痛、儿童交替性偏瘫、快速发作性肌张力障碍帕金森综合征或癫痫。许多已报道的突变会导致功能改变或丧失，而其他一些突变则不会改变功能特性，但会导致蛋白质稳定性降低、蛋白质表达减少或质膜靶向缺陷。钠钾ATP酶经常与其他膜转运体或细胞基质蛋白在特殊的质膜微结构域中组装，但这些相互作用对靶向或蛋白质流动性的影响目前尚不清楚。钠钾ATP酶与锚蛋白B和小窝蛋白-1的既定相互作用基序发生突变，预计会导致质膜靶向改变、定位模式改变以及该酶的扩散行为改变。我们通过荧光相关光谱以及光漂白或光开关后的荧光恢复来监测eGFP标记的钠钾ATP酶构建体在HEK293T细胞质膜中的扩散，研究了这些结合位点突变的后果，并观察到与野生型酶相比存在显著差异，相互作用位点突变组合具有协同效应。这些测量扩展了研究钠钾ATP酶突变后果的可能性，并提供了有关钠钾ATP酶α同工型与细胞基质蛋白、细胞骨架或其他膜蛋白复合物相互作用的信息。
