Prion diseases are fatal neurodegenerative disorders of humans and animals. The prion hypothesis states that PrPSc, a misfolded conformational isoform of the cellular prion protein (PrPC), is the sole component of the infectious particle. Many open questions exist in prion biology including the cellular role of PrPC, the potential involvement of auxiliary factors in prion replication, and the mechanism of PrPSc-induced toxicity in prion disease. The identification of novel prion-like proteins and authentic in vivo prion protein-interacting proteins would certainly assist the process of demystifying these unsolved mysteries. Accordingly, two newly-identified proteins with potential relevance to prion protein biology, Shadoo and DPPX, were selected for biochemical and functional characterization. Shadoo, a hypothetical prion-like protein, is revealed as being a glycoprotein which possesses many overlapping properties with PrPC including neuronal expression, C1-like endoproteolytic processing, and the ability to protect against apoptotic stimuli in cerebellar neurons. Shadoo loosely resembles the disordered N-terminal domain of PrPC and consistent with this notion, Shadoo appears to lack a well-defined structure. Remarkably, Shadoo levels in the brains of mice with clinical prion disease are significantly decreased suggesting that Shadoo may be inherently linked to prion replication or prion disease pathogenesis. These experiments define Shadoo as the third member of the prion protein family and, because of its functional similarities to PrPC, Shadoo may be a useful tool for deciphering the in vivo function of PrPC. DPPX, a neuronal type II transmembrane protein, is demonstrated to be the first protein capable of interacting with all three members of the prion protein family (PrPC, Doppel, and Shadoo) in vivo. Complex formation between prion proteins and DPPX appears to be mediated by multiple binding sites. When coupled with high levels of DPPX expression in cerebellar granular neurons, DPPX is a strong candidate for mediating phenotypic interactions between prion proteins in cerebellar cells. Thus, Shadoo and DPPX comprise two new entry points for studying prion proteins. Further investigation of the roles of Shadoo and DPPX in both the cell biology of prion proteins and prion disease may yield important clues to these enigmatic topics.

Prion diseases are fatal neurodegenerative disorders of humans and animals. The prion hypothesis states that PrPSc, a misfolded conformational isoform of the cellular prion protein (PrPC), is the sole component of the infectious particle. Many open questions exist in prion biology including the cellular role of PrPC, the potential involvement of auxiliary factors in prion replication, and the mechanism of PrPSc-induced toxicity in prion disease. The identification of novel prion-like proteins and authentic in vivo prion protein-interacting proteins would certainly assist the process of demystifying these unsolved mysteries. Accordingly, two newly-identified proteins with potential relevance to prion protein biology, Shadoo and DPPX, were selected for biochemical and functional characterization. Shadoo, a hypothetical prion-like protein, is revealed as being a glycoprotein which possesses many overlapping properties with PrPC including neuronal expression, C1-like endoproteolytic processing, and the ability to protect against apoptotic stimuli in cerebellar neurons. Shadoo loosely resembles the disordered N-terminal domain of PrPC and consistent with this notion, Shadoo appears to lack a well-defined structure. Remarkably, Shadoo levels in the brains of mice with clinical prion disease are significantly decreased suggesting that Shadoo may be inherently linked to prion replication or prion disease pathogenesis. These experiments define Shadoo as the third member of the prion protein family and, because of its functional similarities to PrPC, Shadoo may be a useful tool for deciphering the in vivo function of PrPC. DPPX, a neuronal type II transmembrane protein, is demonstrated to be the first protein capable of interacting with all three members of the prion protein family (PrPC, Doppel, and Shadoo) in vivo. Complex formation between prion proteins and DPPX appears to be mediated by multiple binding sites. When coupled with high levels of DPPX expression in cerebellar granular neurons, DPPX is a strong candidate for mediating phenotypic interactions between prion proteins in cerebellar cells. Thus, Shadoo and DPPX comprise two new entry points for studying prion proteins. Further investigation of the roles of Shadoo and DPPX in both the cell biology of prion proteins and prion disease may yield important clues to these enigmatic topics.

Cardiac accessory β-subunits are part of macromolecular ion channel complexes. They can modulate electrophysiological properties of resulting ion currents and action potentials and are supposed to contribute to cardiac disease e.g. arrhythmias or Brugada syndrome. In my thesis, we characterized the functions of dipeptidyl peptidase-like protein 10 (DPP10), a transmembrane β-subunit of cardiac Na+ and K+ channels. Previous studies revealed that DPP10 is expressed in human heart and acts as regulator of Kv channel kinetics. In electrophysiological experiments, we found that DPP10 modulates Ito through Kv4.3 channel complexes by accelerating current densities and the time course of activation, inactivation and recovery from inactivation. Interestingly, co-expression of DPP10 with Kv4.3 and KChIP2 in CHO cells induced a slowly inactivating fraction of Ito, providing evidence for a contribution of Ito on the sustained outward K+ current in cardiomyoctes. Until then, the sustained fraction of K+ currents was thought to be due to IKur. We further studied the contribution of Kv4-mediated Ito to total K+ currents in human atrial myocytes using 4-Aminopyridine to block IKur in combination with Heteropoda toxin 2 to block Kv4 channels. Using this approach, it was possible to separate an Ito fraction of about 19% contributing to the late current component. These data suggest that the generation of a sustained current component of Ito induced by DPP10 may affect the late repolarization phase of an atrial action potential. To further explore the functions of DPP10, we investigated a potential interaction with Nav channels in cardiomyocytes. It was possible to detect DPP10 in human ventricles, with higher expression levels in patients with heart failure. We demonstrated that DPP10 affects cellular action potentials in isolated rat cardiomyocytes after adenoviral gene transfer indicating a reduction in Na+ current density. Voltage-dependent Na+ channel activation and inactivation curve was shifted to more positive potentials with overexpression of DPP10, resulting in enhanced availability of Na+ channels for activation, along with increasing window Na+ current. Thus, we assumed a role of DPP10 on promotion of arrhythmias via interaction with Nav1.5. The results of this study can help to understand the complex interaction pattern between Nav and Kv channels and the role of their β-subunits, especially DPP10. In conclusion, DPP10 was identified as a new modulator of Kv and Nav currents in the human heart, suggesting that this β-subunit may contributes to cardiac arrhythmias and might be a new therapeutic target.:1 Introduction 1.1 The cardiac action potential 1.2 Cardiac potassium channels 1.2.1 The Kv4.3 channel complex 1.2.2 Accessory β subunits of K+ channel 1.2.3 The Kv1.5 channel 1.2.4 Separation of Ito and IKur in native cardiomyocytes 1.3 Cardiac sodium channels 1.3.1 Molecular construction of Nav1.5 channel 1.3.2 Accessory β subunits of Na+ channel 1.3.3 The role of Nav1.5 in cardiac electrical disorders 1.4 Aim of the thesis and systematic approach 2 The research articles 3 Summary 4 Zusammenfassung 6 References 7 Appendices 7.1 Abbreviations