Analysis of a co-endocytosis of Na/K ATPase with TASK-3 via biotin labelling

The Na/K ATPase was identified for the phosphorylated TASK-3 C-terminus from cardiac tissue by MS analysis. In addition, proteins which are involved in endocytosis processes were identified for the phosphorylated TASK-3 C-terminus as well as different 14-3-3 isoforms (Figure 33). Efendiev et al., published that 14-3-3 is important to recruit phosphoinositide 3-kinase to the Na/K ATPase during clathrin-dependent endocytosis (Efendiev et al., 2005). This prompted the idea, that 14-3-3 dimers may bring phosphorylated TASK-3 and the Na/K ATPase in close proximity for a potential co-endocytosis mediated by AP-1 and dynamin. To further investigate this hypothesis, I performed an affinity purification experiment from rat heart lysate in a similar fashion to the one for subjected to MS. The affinity purification experiment was performed without 14-3-3 inhibition followed by incubation of phosphorylated TASK-3 protein variants and Western blotting analysis with 14-3-3 and Na/K ATPase antibodies (Figure 34). 14-3-3 was strongly enriched with the phosphorylated TASK-3 WT protein but not with the unphosphorylated controls (Figure 34). However, no Na/K ATPase was detected with any of the TASK-3 mutant proteins (Figure 34). Altogether, I was unable to confirm an interaction between the phosphorylated TASK-3 C-terminus and Na/K ATPase by Western blot. This indicates that the binding affinity between TASK-3 and Na/K ATPase is below the affinity of Western blot quantification and shows that MS analysis has a much higher sensitivity than Western blotting (Bass et al., 2017).

Chapter IV: Identification of novel interaction partners for TASK channels in the heart

Figure 34. Western blot detection of the Na/K ATPase in samples obtained by TASK-3 pull-down experiments with heart lysate.

Analysis of affinity purification of TASK-3 protein variants with rat heart lysate and no R18 inhibitor treatment. The interaction between TASK-3 and 14-3-3 as well as with the Na/K ATPase were analysed (lower panel) by Western blot.

Verification of a potential Na/K ATPase interaction with TASK-3 was not possible with pull-down experiment followed by Western blot analysis. To further investigate the hypothesis of a potential co-endocytosis between TASK-3 and the Na/K-ATPase, the endocytosis rate of the Na/K ATPase was stimulated in rats. Therefore, whole rat hearts were stimulated with the putative endocytosis enhancing drug ouabain. Ouabain is a cardiac glycoside, which inhibits the Na/K-ATPase pump and was proposed to induce endocytosis in the heart (Liu, 2005). Internalization of endocytosed surface proteins was monitored by subsequent surface biotinylation of the hearts. The described treatment was performed in the context of Langendorff perfusion experiments. Rat hearts were perfused with different concentrations of ouabain followed by biotinylation with non-cell-permeable biotin to label proteins which are exposed at the cell surface (Figure 35A). Biotin-perfusion was performed at 4°C to stop intracellular trafficking during the labelling. Rat hearts were used since the TASK-3 Na/K ATPase interaction was identified in rat. However, the Na/K ATPase has a reduced sensitivity towards ouabain in rats (Cherniavsky-Lev et al., 2014).

To ensure, that ouabain induces endocytosis in rat hearts a low dose of 100 nM and a high dose of 50 µM were used. To control the experiment and monitor background biotinylation the hearts were perfused in 4 different groups: (1) No ouabain and no biotin treatment during perfusion, (2) no ouabain but biotin treatment, (3) 100 nM ouabain and biotin treatment and (4) 50 µM ouabain and biotin treatment.

After the heart treatment using Langendorff perfusion, membranes and cytosol were fractionated and analysed for the amount of biotinylated proteins by Western blotting with

anti-14-3-3 pan

Chapter IV: Identification of novel interaction partners for TASK channels in the heart ouabain and biotin, no enrichment of biotinylated proteins was visible for membranes or cytosol (Figure 35B). Biotinylated proteins were detected in membranes from heart samples which were perfused with biotin, but no differences were visible due to ouabain treatment (Figure 35B).

Surprisingly, biotinylated proteins were also enriched from the cytosol fraction, even though the amount of enriched proteins was reduced in comparison with the membrane fractions analysed (Figure 35B).

Figure 35. Enrichment of biotinylated proteins from rat hearts treated with ouabain.

(A) Rat hearts were treated in a Langendorff perfusion with ouabain (100 nM or 50 µM) followed by surface biotinylation with non-membrane permeable Sulfo-NHS-LC-biotin at 4°C. (B) Membranes or cytosol separated from the Langendorff perfused hearts were analysed for the enrichment of biotinylated proteins. Inputs, flow throughs and elutions were analysed by Western blotting with fluorophore-conjugated streptavidin.

Chapter IV: Identification of novel interaction partners for TASK channels in the heart Biotinylated proteins were enriched from the cytosolic fraction of hearts which were treated with biotin and ouabain (Figure 36). To exclude cross-contamination of the sample during the subcellular fractionation, cytosolic and membrane protein markers were analysed by Western blot using GAPDH and Na/K ATPase antibodies respectively. The cytosolic protein GAPDH was only detected in cytosol samples whereas Na/K ATPase was only detected in the membrane fraction samples (Figure 36). This indicates that the separated fractions of membrane or cytosol were clean, but it does not exclude that small amounts of biotin were introduced into the cytosol during sample preparation.

Figure 36. Probing for different marker proteins in ouabain treated heart membrane and cytosol.

The purity of the separated membrane or cytosolic fraction was confirmed by Western blotting and the detection of the cytosolic marker protein GAPDH (upper panel) or the membrane protein Na/K ATPase (lower panel).

4.3.1.4 Enrichment of Na/K ATPase and 14-3-3 in biotinylated heart