No / Voltage clamp was used to investigate the effects of N-[2-p-bromo-cinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), a potent inhibitor of PKA, on transient outward K+ current (Ito) and inward rectifying K+ current (IK1) in rat cardiac muscle. Initial experiments, performed using descending voltage ramps, showed that H-89 inhibited both the outward and inward ramp currents in a concentration-dependent manner at concentrations between 5 and 60 ¿mol l¿1. A similar degree of inhibition was observed when Ito and IK1 were recorded using square wave depolarising and hyperpolarising voltage steps, respectively. The IC50 was 35.8 ¿mol l¿1 for Ito and 27.8 ¿mol l¿1 for IK1 compared to 5.4 ¿mol l¿1 for L-type Ca2+ current (ICa). The Hill coefficients for Ito, IK1 and ICa were ¿1.97, ¿1.60 and ¿1.21, respectively. In addition to inhibiting Ito amplitude, H-89 also accelerated the time to peak and the rate of voltage-dependent inactivation so that the time course of Ito was abbreviated. Paired-pulse protocols were performed to study the effects of H-89 on steady-state activation and inactivation as well as recovery from voltage-dependent inactivation. H-89 produced a concentration-dependent rightward shift in voltage-dependent activation but had no significant effect on steady-state inactivation. Recovery from voltage-dependent inactivation was delayed, although this was only visible at the highest concentration (60 ¿mol l¿1) used. In experiments investigating the effects of elevated cyclic AMP, the ß-adrenergic agonist isoprenaline and the phosphatase inhibitor calyculin A had no major effects on Ito or IK1. Data suggest that the effects of H-89 on K+ currents are more complex than simple inhibition of PKA-mediated phosphorylation.

Ion channels play a critical role in maintaining homeostasis by moving various ions in and out of cells. The Na+-K+-2Cl- or NKCC1 ion channel is involved in the regulation of Na+, K+, and Cl- across cell membranes, and plays a key role in many forms of cellular physiology. In the cochlea, NKCC1 is involved in endolymph production and maintenance of the endocochlear potential. Our hypothesis is that blocking NKCC1 channels should directly impact auditory sensitivity causing hearing loss. Our lab has also shown that the hormone aldosterone (ALD) can upregulate NKCC1 protein expression in vitro and in vivo. In the present investigation, we use electrophysiology and molecular biology techniques to study the biophysical mechanisms underlying the action of ALD in vitro on NKCC1 in the SH-SY5Y cell line. Our initial protein expression studies using RT-PCR found that proteins specific to NKCC1channels were present in SH-SY5Y neuronal cells. Whole cell currents measured using patch clamp methodology, were used to analyze the effects of various compounds on NKCC1 in the SH-SY5Y cell line. Control data were collected under perfusion of extracellular solution (ECS), then ECS containing 10µM bumetanide was applied, and, finally a washout condition completed the experiment. Similar experiments were conducted using ALD, and we observed an increase in K+ currents when bumetanide as well as when ALD was applied. This is the first report that indicates that ALD can directly regulate K+ channels in SH-SY5Y cells.

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