PT  - JOURNAL ARTICLE
AU  - Cornelia Junghans
AU  - Vladana Vukojević
AU  - Neslihan N. Tavraz
AU  - Eugene G. Maksimov
AU  - Werner Zuschratter
AU  - Franz-Josef Schmitt
AU  - Thomas Friedrich
TI  - Disruption of ankyrin B and caveolin-1 interaction sites alters Na<sup>+</sup>,K<sup>+</sup>-ATPase lateral diffusion in HEK293 cell plasma membranes
AID  - 10.1101/141291
DP  - 2017 Jan 01
TA  - bioRxiv
PG  - 141291
4099  - http://biorxiv.org/content/early/2017/05/23/141291.short
4100  - http://biorxiv.org/content/early/2017/05/23/141291.full
AB  - 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 (FHM2), Alternating Hemiplegia of Childhood (AHC), Rapid Dystonia Parkinsonism (RDP) 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. Mutational disruption 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 living HEK293T cells by fluorescence correlation spectroscopy (FCS) as well as fluorescence recovery after photobleaching (FRAP) or photoswitching (FRAS) 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 α2-isoform with cellular matrix proteins, the cytoskeleton or other membrane protein complexes.