PLN disease mutations are localized in the ER in mammalian cells

In parallel to the investigation of the PLN R14del mice disease models, expression and localization of PLN mutant proteins in mammalian cell lines were investigated. Protein expression was analysed by Western blotting in HEK293T and HeLa cells. The expression levels of the two human disease mutants were found to be reduced by Western blotting in both cell lines. The PLN-opsin mutant proteins did not form SDS-resistant pentamers in HEK or HeLa cells (Figure 25). Pentamer formation is achieved by the transmembrane domain of PLN (Kimura et al., 1997), which is present in these constructs. Perhaps the opsin-tag prevented pentamer formation. Overall, there was no difference visible in HEK293T or HeLa cells regarding the expression of the PLN constructs (Figure 25).

Localization analysis of the PLN mutant variants in mammalian cells demonstrated the correct localization of all PLN mutants to the ER compartment by co-staining with the ER marker Sec61beta (Figure 26). Interestingly, also the PLN R14del mutation was identified in the ER by co-staining with SEC61beta. In the literature the PLN R14del mutation has been reported to be localized at the plasma membrane after expression in HEK cells (Haghighi et al., 2012). I have not been able to confirm this result in my experimental set up.

In addition to ER localization, different cellular compartments were investigated such as early and late endosomes. If the PLN disease mutants would be misrouted to the plasma membrane, they should be also visible at some point in the endosome, since endosomal pathways receive all incoming material from the plasma membrane and serve as an initial sorting route for either recycling or degradation (Scott et al., 2014). I performed IF experiments with early and late endosomal marker proteins and the opsin-tag fused to PLN mutants were performed. No localization of any PLN mutant could be observed in either early nor late endosomal vesicles. The PLN R14del mutation was also not localized in endosomal vesicles (Figure 227,28). Altogether, I could not confirm mislocalization of the R14del PLN mutation expressed in mammalian cells. Taken together with the results from

Chapter III: Further analysis of the PLN/14-3-3 interaction homozygous and heterozygous PLN R14del mouse models, where barely any R14del PLN protein could be identified, my data support the already suggested hypothesis of PLN R14del aggregate formation (te Rijdt et al., 2016) rather than mislocalization. To further proof this hypothesis, cardiac myocytes should be isolated from the homozygous and heterozygous PLN R14del mouse model and the staining experiments should be repeated.

In addition, my IF analysis supports published work from Butler et al who also did not observe any PLN localization in early endosomes (Butler et al., 2007). Interestingly, Butler et al identified PLN in the ERGIC and suggested a model where PLN is maintained in the ER by retrieval trafficking between ERGIC and ER (Butler et al., 2007). To further confirm the PLN localization in the ERGIC a corresponding IF experiment should be performed. In addition, it seems worthy to also repeat the experiment with an alternative tag, such as a myc-tag to exclude that the opsin-tag is influencing the ability of PLN to form pentamers and thereby alter the localization. In addition, it would be interesting if phosphorylation of PLN in mammalian cells influences the localization. Therefore, cells could be stimulated with isoprenaline to activate beta-adrenergic stimulation.

3.4.4 14-3-3 binds PLN pentamers in human atria

Most of the analysis of the 14-3-3/PLN interaction was performed with in-vitro approaches or in mouse models. Although PLN is very conserved in human and mouse (McTiernan et al., 1999) differences between both species were observed. PLN deletion in mice shows increased cardiac contractile function without the development of HF (Haghighi et al., 2003). In humans all PLN mutations that lead to protein truncation are lethal due to dilated cardiomyopathy and subsequent HF (Haghighi et al., 2003).

As a consequence, it is important to understand the differences in cardiac physiology and Ca²⁺ handling in mice and humans. The heart rate in mice is 10 times faster than in humans. Moreover, 92% of Ca²⁺ removal is achieved by SERCA in mice, meanwhile just 70% of Ca²⁺ is transported by SERCA in humans. In addition the basic ventricular motor proteins responsible for cardiac contraction differ in mice and human (Haghighi et al., 2003). With a 14-3-3 pull-down experiment and cardiac membranes separated from human atrial biopsy samples I enriched PLN pentamers (Figure 29). This verifies a physical 14-3-3/PLN interaction in human atrium. Atria and ventricle show different characteristics such as myocardial mass or wall thickness (Filgueiras-Rama & Jalife, 2016). In addition, they show functional differences in intracavitary pressures, ion channel composition, and electrophysical characteristics (Filgueiras-Rama & Jalife, 2016). As a consequence, it is important to also investigate the 14-3-3/PLN interaction in human ventricular samples.

Chapter III: Further analysis of the PLN/14-3-3 interaction

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

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

4.1 Introduction

The family of K2P channels is one out of three K⁺ ion channel families involved in cardiac action potential generation and electrical signal transduction. K2P channels are background leak channels for potassium currents which are important regulators of the resting membrane potential (Hughes et al., 2017). The K2P channel family is defined by a structure of four transmembrane domains and two-pore forming domains per monomer (Goldstein et al., 2005). K2P channels are strongly expressed in the brain and central nervous system (Lesage, 2003). However, several members of K2P channels also were identified to be expressed in the heart like TASK channels (Gurney & Manoury, 2009).

TASK channels are one group within the K2P channel family, defined by their high sensitivity to extracellular acidic stress (Duprat et al., 1997; Talley et al., 2000). Three different channels belong to the group of TASK channels: TASK-1, TASK-3 and the silent TASK-5 channel. TASK-1 is expressed on mRNA and protein levels in the heart and mostly in the atrium. Whereas for TASK-3, only mRNA transcripts could be found in the heart and in the aorta but was absent based on protein levels (Gurney & Manoury 2009).

Homo- and heterodimeric TASK channels have been identified within the same channel family but also within different K2P channel families. For the TASK-1/TASK-3 heterodimeric channel different electrophysical and pharmacological properties were identified in comparison with the corresponding homodimers (Czirják & Enyedi, 2002). In 2007, Putzke et al detected first TASK-1 like currents in rat ventricular myocytes. They reported that the TASK-1 current contributes substantially to the total outward current during the plateau phase of the action potential (Putzke et al., 2007) indicating the physiological importance.

In line, TASK homo- and heterodimers have been shown to be physiologically relevant in various tissues (Gurney & Manoury 2009). Interestingly, Rinné et al suggested the presence of a functional heteromeric TASK-1/TASK-3 channel in hearts (Rinné et al., 2015). Altogether, TASK channels have been reported as an important regulator of the action potential duration and for resetting the resting membrane potential in the mammalian cardiovascular system.

Functional TASK channels need to be exposed at the cell surface. Hence, trafficking of TASK channels is tightly regulated. The TASK-1 and TASK-3 proteins are highly conserved except for the cytosolic C-terminal domain which shows only 34% homology (O'Kelley et al., 2015, Rajan et al., 2002). The last 15 AA of the C-terminus of TASK-1 as well as TASK-3 channels contain a so called “trafficking control region” with several

Chapter IV: Identification of novel interaction partners for TASK channels in the heart (Figure 30) (Kilisch et al., 2015). The trafficking control region of TASK-1 and TASK-3 include an ER retrieval sequence (KRR), as well as an overlapping 14-3-3 binding motif containing one phosphorylatable serine residue (Zuzarte et al., 2009). Several binding partners are already known to interact with the TASK C-terminus, like the COPI vesicular coat as well as the phospho-adaptor protein 14-3-3. Binding of COPI or 14-3-3 to the TASK channel is regulated by PKA-dependent phosphorylation of the C-terminal trafficking control region (Mant et al., 2011). The phosphorylated serine residue S393 in TASK-1 and S373 in TASK-3 bind 14-3-3 with high affinities. 14-3-3 binding sterically hinders COPI interaction, promoting forward trafficking of the channels to the cell surface (Kilisch et al., 2016). After 14-3-3 binding the TASK channels leave the early secretory pathway on their way to the cell surface, where they get glycosylated. Glycosylation is an important step for correct surface expression of many ion channels as shown for KATP or Kv1.4 (Steele et al., 2007). COPI binding initiates retrograde trafficking of the channels to the ER and cis-Golgi (together also called “early secretory pathway”) thus allowing for correct assembly prior to cell surface expression of the channel (Mathie, 2007).

Interestingly, the TASK-1 channel has a second serine residue in the trafficking control region adjacent to the 14-3-3 binding S393 (Figure 30), which is absent in the TASK-3 channel. It was shown that the second serine residue (S392) in TASK-1 can also be targeted by PKA in vitro and in vivo (Kilisch et al., 2016). The S392 residue was found to be an inhibitory residue for 14-3-3 binding upon phosphorylation (Kilisch et al., 2016).

Presence of the phosphorylated S392 residue in the TASK-1 C-terminus significantly reduced the affinity for 14-3-3. Consequently, the surface expression of TASK-1 was also strongly reduced upon S392 phosphorylation (Kilisch et al., 2016). Although TASK-1 and TASK-3 channels are very conserved a couple of protein interaction partners were exclusively identified for TASK-1. The cytosolic adaptor protein p11 binds TASK-1 via a 20 aa long stretch in the C-terminus, which is also called p11 binding site and is located upstream over the trafficking control region (Figure 30). The TASK-1 p11 interaction leads to the retrieval of the channel to intracellular compartments of the early secretory pathway (Renigunta et al., 2006). Moreover, it was shown that TASK-1 interacts with the endosomal SNARE protein syntaxin-8. Interestingly, the interaction which syntaxin-8 and TASK- 1 was found to be involved in endocytosis processes (Renigunta et al., 2014).

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

Figure 30. Schematic representation of TASK-1 and TASK-3.

Both channels show high sequence homology except for the C-terminus. The trafficking control region is represented in yellow (14-3-3 binding motif) and blue (ER retention/retrieval motif). The i20 domain is shown in green and a potential internalization motif is displayed in red (aa 294-314).

Compared to TASK-1 (two adjacent serine residues, S392, S393), TASK-3 possesses a lysine residue adjacent to the serine residue, which is part of the 14-3-3 binding motif. The 14-3-3 phospho-binding residue in the TASK-1 (S393) and TASK-3 (S373) trafficking control domain is depicted in red. Figure adapted from (Kilisch et al., 2015).

Endocytosis can mediate retrieval from the cell surface, recycling, and degradation of membrane proteins. Therefore, endocytosis is also important to regulate cell surface expression of TASK channels (Mant et al., 2013). Clathrin-dependent endocytosis is one of the best investigated endocytosis pathways and involves adaptor proteins like AP-2, which direct the cargo protein into the vesicle (Lafer, 2002). It was already shown that TASK-1 and TASK-3 co-localize with clathrin. The co-localization was strongly decreased after disruption of the clathrin-mediated pathway (Gabriel et al., 2012; Mant et al., 2013).

However, for TASK-3 it was shown that endocytosis is happening more rapidly than for

Chapter IV: Identification of novel interaction partners for TASK channels in the heart with proteins involved in the clathrin-dependent endocytosis, the syntaxin-8 interaction was only identified with TASK-1. This interaction was found to initiate co-endocytosis of both proteins together in a cooperative manner (Renigunta et al., 2014). It was suggested that the C-terminus of TASK-1 and the N-terminus of syntaxin-8 bind AP-2, which initiates clathrin-dependent co-endocytosis. A potential benefit resulting from the co-endocytosis could be that syntaxin-8 may influence the final destination of the vesicle by the interaction with SNARE proteins localized in different compartments (Renigunta et al., 2014). In addition it has been reported that PKC is involved in TASK-1 regulated endocytosis and that another endocytosis motif (SRERKLQYSIP) very close to the p11 binding motif in the TASK-1 C-terminus is involved in this process (Gabriel et al., 2012). The authors showed reduced TASK-1 currents after PKC stimulation in mammalian cells and an increased TASK-1 internalization and intracellular puncta formation by microscopy analysis.

Interestingly, Gabriel et al suggested that PKC phosphorylation of the second serine (S324) in the identified endocytosis motif (SRERKLQYSIP) of TASK-1 may allow 14-3-3 binding and subsequent endocytosis.

The function and regulation of TASK channels in the heart is still in its infancy and not much is known about cardiac mediators, which may be involved in this regulation. The fact, that more interaction partners were identified for TASK-1 and the stronger expression in heart, indicate that TASK-1 channels are regulated in a more complex way. In line with this notion, the TASK-1 channel has a second PKA phosphorylatable serine residue (S392) in the trafficking control region which function is not fully understood so far. The goal of this chapter was the analysis of the cardiac interactome for the extreme C-terminus of TASK-1 and TASK-3, including the trafficking control domain. Phosphorylation is an important regulator of TASK channels thereby the analysis was performed differentiated between the phosphorylated or unphosphorylated status of the C-terminus of the channels.

Chapter IV: Identification of novel interaction partners for TASK channels in the heart 4.2 Material and Methods