名古屋大学博士学位論文 学位の種類 : 博士(医学)(課程) 学位授与年月日:平成10年6月4日 李鍾國氏の博士論文として提出された

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<p>The neural code from the inner ear to the brain is dynamically controlled by central nervous efferent feedback to the audio-vestibular epithelium. Although such efference provides the basis for a cognitive control of our hearing and balance, we know surprisingly little about this feedback system. This project has investigated the applicability of a transgenic mouse model, expressing a fluorescent protein under the choline-acetyltransferase (ChAT) promoter, for targeting the cholinergic audio-vestibular efferent neurons in the brainstem. It was found that the mouse model is useful for targeting the vestibular efferents, which are fluorescent, but not the auditory efferents, which are not highlighted. This model enables, for the first time, physiological studies of the vestibular efferent neurons and their synaptic inputs. We next assessed the expression of the potassium channel family Kv4, known to generate transient potassium currents upon depolarization. Such potassium currents are found in auditory efferent neurons, but it is not known whether Kv4 subunits are expressed in these neurons. Moreover, it is not known if Kv4 is present and has a function in the vestibular efferent neurons. Double labelling with anti-ChAT and anti-Kv4.2 or Kv4.3 demonstrates that the Kv4.3 subunits are abundantly expressed in audio-vestibular efferents, thus indicating that this subunit is a large contributor to the excitability and firing properties of the auditory efferent neurons, and most probably also for the vestibular efferent neurons. In addition, we also unexpectedly found a strong expression of Kv4.3 in principal cells of the superior olive, the neurons which are important for sound localization.</p>

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

The neural code from the inner ear to the brain is dynamically controlled by central nervous efferent feedback to the audio-vestibular epithelium. Although such efference provides the basis for a cognitive control of our hearing and balance, we know surprisingly little about this feedback system. This project has investigated the applicability of a transgenic mouse model, expressing a fluorescent protein under the choline-acetyltransferase (ChAT) promoter, for targeting the cholinergic audio-vestibular efferent neurons in the brainstem. It was found that the mouse model is useful for targeting the vestibular efferents, which are fluorescent, but not the auditory efferents, which are not highlighted. This model enables, for the first time, physiological studies of the vestibular efferent neurons and their synaptic inputs. We next assessed the expression of the potassium channel family Kv4, known to generate transient potassium currents upon depolarization. Such potassium currents are found in auditory efferent neurons, but it is not known whether Kv4 subunits are expressed in these neurons. Moreover, it is not known if Kv4 is present and has a function in the vestibular efferent neurons. Double labelling with anti-ChAT and anti-Kv4.2 or Kv4.3 demonstrates that the Kv4.3 subunits are abundantly expressed in audio-vestibular efferents, thus indicating that this subunit is a large contributor to the excitability and firing properties of the auditory efferent neurons, and most probably also for the vestibular efferent neurons. In addition, we also unexpectedly found a strong expression of Kv4.3 in principal cells of the superior olive, the neurons which are important for sound localization.