Enrichment of Na/K ATPase and 14-3-3 in biotinylated heart membranes . 163

I was able to show that membrane proteins expressed at the cell surface can be visualized with biotin surface labeling. Next, I wanted to analyse the behavior of biotinylated Na/K ATPase with or without ouabain induced endocytosis. Therefore, enriched biotinylated proteins from the membrane fraction were probed for the amount of Na/K ATPase under different ouabain concentrations. Ouabain has been shown to initiate Na/K ATPase endocytosis (Cherniavsky-Lev et al., 2014) and therefore ouabain-treated heart membranes should show less biotinylated Na/K ATPase after enrichment with neutravidin pull-downs. In the elution of the neutravidin pull-down, no biotinylated proteins were detected in the control sample which was not treated with biotin or ouabain. Biotinylated Na/K ATPase was enriched in all samples perfused with biotin, but no systematic

Chapter IV: Identification of novel interaction partners for TASK channels in the heart visible among the different ouabain concentrations (Figure 37A,B). Altogether, the enrichment of proteins which are expressed at the cell surface from complete rat hearts was possible with the help of neutravidin enrichment, but no significant difference was visible for the amounts of Na/K ATPase present at the cell surface after ouabain treatment.

Figure 37. Biotinylated Na/K ATPase protein was enriched in biotinylated heart membranes.

(A) Na/K ATPase protein levels in perfused heart samples treated with different concentrations of ouabain and biotin were analysed. Biotinylated proteins were purified with neutravidin resin and analysed by Western blot with a Na/K-ATPase antibody. (B) Quantification of experiment shown in (A) using values of the Na/K ATPase intensity normalized to the sample perfused with biotin but without ouabain. The data of three independent experiments are presented as mean ± SEM. An unpaired t-test showed significant differences between control versus biotin (***p=0.0003), but not between biotin versus ouabain treated samples.

Ouabain treatment did not influence the surface expression of Na/K ATPase in membranes. A potential co-endocytosis of the Na/K- ATPase with TASK-3 may require 14-3-3 which clamps the proteins in close proximity. To further investigate the role of 14-3-3, enriched biotinylated membrane proteins from neutravidin affinity experiments of the ouabain treated hearts were analysed regarding the presence of 14-3-3 proteins by Western blot. In the input samples used for the neutravidin enrichment experiment no 14-3-3 was detected at all. However, after neutravidin enrichment weak signals for 14-3-3

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Chapter IV: Identification of novel interaction partners for TASK channels in the heart were detected (Figure 38). These 14-3-3 proteins were bound to biotinylated membrane proteins. In line, no 14-3-3 was detected in the control sample which was not treated with biotin (Figure 38). The amount of 14-3-3 proteins detected from biotinylated membranes did not vary between hearts treated with different doses of ouabain (Figure 38).

Figure 38. 14-3-3 was enriched in biotinylated heart membranes.

(A) Analysis of 14-3-3 protein levels in heart membranes treated with different concentrations of ouabain. Biotinylated proteins from perfused rat hearts were purified with neutravidin and analysed with Western blot and a 14-3-3 antibody. (B) Quantification of experiment shown in (A) using values of the 14-3-3 intensity normalized to the sample just perfused with biotin but without ouabain. The data of three independent experiments are presented as mean ± SEM. An unpaired t-test showed significant differences between control versus biotin (*p=0.0122), but not between biotin and ouabain treated samples.

4.3.1.5 Enrichment of glycosylated Na/K ATPase in rat heart membranes Glycosylation is one of various post-translational modifications to ensure correct trafficking of proteins within the cell. Glycosylated proteins are transported to the plasma membrane (Bas et al., 2011). For presenting the membrane proteins to the extracellular matrix glycosylation is required as well (Hart, 1992). In addition, it was reported that glycosylation protects proteins from degradation resulting in a reduced rate of endocytosis (Altschuler et al., 2000; Russell et al., 2009). Ouabain treatment showed no influence on the amount of Na/K ATPase present in the plasma membrane or the amounts of 14-3-3 interacting with cell surface proteins. Another method to test whether ouabain influences the amount of cell-surface expressed membrane proteins is the analysis of glycosylated proteins. In order to test the effect of ouabain on Na/K ATPase endocytosis I analysed the glycosylation status of the Na/K ATPase with wheat germ agglutinin (WGA) enrichment experiments.

WGA resin efficiently binds glycosylated proteins (Goldstein et al., 1997). Therefore, WGA kDa 34

Chapter IV: Identification of novel interaction partners for TASK channels in the heart analysed with Coomassie stained SDS-PAGE (Figure 39). For heart membranes the amount of glycosylated proteins in the elution is reduced in comparison with the input or flow-through fraction. Moreover, slightly less glycosylated proteins were eluted after WGA affinity enrichment from the ouabain treated membrane samples. But no difference between low and high ouabain concentrations were observed.

Figure 39. Glycosylated proteins were enriched from rat heart membranes.

Affinity enrichment of glycosylated proteins from ouabain treated cardiac membranes with WGA resin. Enrichment of glycosylated proteins was analysed on Coomassie stained SDS-PAGE. Inputs, flow throughs and elutions of the purification were analysed.

The analysis of the amount of glycosylated proteins in ouabain treated heart samples showed slightly less glycosylated proteins enriched from heart membranes after ouabain treatment. This result indicates that less membrane proteins are exposed to the extracellular matrix after ouabain treatment and may allow the conclusion that the endocytosis rate was increased in these samples. However, this effect should be also visible for the Na/K ATPase. Therefore, the samples were probed for specific amounts of glycosylated Na/K ATPase by Western blot with a Na/K ATPase antibody (Figure 40A).

Input samples from solubilized membranes for the different ouabain and biotin treatments were compared with flow throughs and elutions after WGA affinity purification (Figure 40A).

Glycosylated Na/K ATPase was massively enriched with the WGA affinity purification (Figure 40A). No statistical differences for Na/K ATPase protein levels were detected for the control heart samples in comparison with the different ouabain treated samples (Figure 40B).

Chapter IV: Identification of novel interaction partners for TASK channels in the heart Altogether, the Na/K ATPase pump was strongly enriched in membranes after WGA enrichment purification, indicating that – as expected – Na/K ATPase is a major cardiac membrane glycoprotein, but no differences were detected for the ouabain treatment.

Figure 40. The Na/K ATPase was glycosylated in ouabain treated and control heart membranes.

(A) Glycosylated proteins were affinity purified with WGA, the elution was analysed by Western blotting with a Na/K ATPase antibody. Different levels of ouabain and different time points of the affinity purification were compared. (B) Quantification of experiment shown in (A) using values of the Na/K ATPase intensity normalized to the control sample which was not perfused with biotin or ouabain. The data of three independent experiments are presented as mean ± SEM. An unpaired t-test showed no significant differences between the samples.

4.3.1.6 Na/K ATPase and many other membrane proteins were biotinylated