This dissertation, "Structure-function and Physiological Properties of HCN-encoded Pacemaker Channels" by Kai, Wang, 王凱, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled Structure-Function and Physiological Properties of HCN-encoded Pacemaker Channels Submitted by Wang Kai For the degree of Doctor of Philosophy at the University of Hong Kong In August, 2007 Pacemaker current I, encoded by the hyperpolarization-activated, cyclic nucleotide-gated (HCN) gene family, contributes significantly to cardiac pacing. As a result, it has become an intensively investigated target to modulate heart rate and function by using pharmacological, genetical and cell-based methods. However, detailed structure-function relationships of HCN channel and physiological roles of I current in different cell types remain largely obscure. On the other hand, other components, such as inward rectifier current (I ) has been proposed to play a more K1 important role in pacemaker activity. Firstly, to further explore the functional role of I current in pacemaking, I compared the contribution of I and I current to the automaticity of cultured neonatal rat f K1 ventricular myocytes (NRVMs). I found adenovirus mediated genetically engineered HCN1 channel overexpression can recover the automaticity of quiescent NRVMs, confirming the crucial role of I as the initiator of the pacemaker activity. I and f K1 iiiprobably sodium current but not I are responsible for the time-dependent change of automaticity. Furthermore, other mechanisms, such as connexin-encoded gap junction may also plays an important role in maintaining the synchronized electrical activity of NRVM monolayer culture. These findings reveal the complexity of cardiac pacemaker mechanism, and suggest future efforts should be paid to explore the strategy of fine-tuning I -induced pacemaking activity. Secondly, to study the structure-function relationship of HCN channel blocking, the functional consequences of alanine-scanning mutagenesis in pore-forming region were studied. Pharmacological and kinetics assays showed that alanine-substitutions of several residues in the middle of S6 segment of HCN1 channel significantly alter both voltage-dependence of activation and sensitivity to the specific blocker ZD7288. Based on these results, I proposed that these residues form a hydrophobic binding pocket within the activation gate for ZD7288. These findings provide useful information to develop more specific HCN blockers and genetically engineered HCN channels for clinical application. Finally, the electrophysiological properties of pluripotent human and mouse embryonic stem cells (ESCs), which can differentiate into pacemaker cells, were characterized. Interestingly, functional expression of I current was detected in mouse but not human ESCs. In addition, several specialized ion channels are expressed at the mRNA and functional levels in both pluripotent mouse and human iv+ ESCs. The main component, depolarization-activated delayed rectifier K currents (I ) were demonstrated to play an important role in controlling the cell KDR proliferation. By contrast, neither voltage gated sodium nor calcium currents were detected in both cells. Microarray and RT-PCR analyses identified several candidate genes for the ionic currents discovered. These findings provide insights into the similarities and differences between the two species and offer a possible strategy to inhibit or eliminate tumorgenicity of residual pluripotent