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Molecular mechanism of cyclic nucleotide action on HCN pacemaker channels Ng, Leo Chun Ting

Abstract

HCN pacemaker channels generate the “funny” current that is responsible for initiation and regulation of electrical activity in the heart and the nervous system. Activated near resting membrane potentials, the channel conducts a net inward current that maintains action potential. The direct binding of cyclic AMP upon adrenaline release also enhances channel opening, leading to an increased frequency of action potential. The cytoplasmic cAMP-binding domain is coupled to the C-linker found between the binding domain and the transmembrane domain, subsequently triggering the opening of the pore. Despite extensive research, the mechanism that links the binding event to channel facilitation is still unclear. In this thesis, we looked into the different aspects of the missing link. In addition to basic understanding, the significance of learning about HCN channel modulation by cAMP is that there may be therapeutic advantage of controlling heart rate via drug interaction with the cyclic nucleotide binding pocket. Using the isolated C-linker/binding domain, we pinpointed residues in the binding pocket that contribute to strong affinity and ligand specificity by single residue alanine-scanning and isothermal titration calorimetry for measurement of binding affinity. By comparing our binding data to functional measurements of potency in full-length channels containing the same single substitutions, we found that two residues, L633 and I636, reduced potency more severely than affinity when mutated, and proposed that these residues are involved in a post-binding transition event. We also found that two partial agonists, cCMP and cIMP, bound to the canonical site, but failed to fully promote tetrameric gating ring which is found on the inner side of the pore and hypothesized to facilitate its opening. We proposed that the weakened interactions between the partial agonists and the C-helix of the binding domain limit the formation of the gating ring and led to reduced facilitation of opening in the full-length channel. Finally, we elucidated the mechanism behind two disease-associated mutations found in the cytosolic C-linker/binding domain portion of the HCN2 and HCN4 channels to gain a better understanding of how they influence cAMP binding and channel opening, and cause epilepsy and profound bradycardia, respectively.

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Attribution-NonCommercial-NoDerivatives 4.0 International

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