UBC Theses and Dissertations
Structural and functional consequences of a sinus tachycardia-associated mutation on the regulation of pacemaker channel by cyclic nucleotides Xia, Joanna
The “funny” (If) current, which is generated by HCN (Hyperpolarization-activated cyclic nucleotide-gated channels), contributes to the diastolic depolarization phase of the action potential. Regulation of If by cAMP represents a key mechanism by which heart rate is autonomically regulated. HCN channels open more easily when cyclic nucleotides (cAMP or cGMP) bind to a domain in the intracellular C-terminus in each of the four identical subunits. Disease-associated mutations in HCN often interfere with the regulation of HCN by cAMP. Therefore, understanding how the cAMP regulation is perturbed in the context of diseases is the first step to developing targeted therapies and treatment. Here, we analyze the effect of the first discovered gain-of-function mutation, R524Q, in HCN isoform 4. This mutation is found in a region called the C-linker that connects the cyclic nucleotide-binding domain to the pore. Previously published concentration-response curves collected from electrophysiology experiments show that R524Q results in increased cAMP potency, as seen from a shift of the concentration-response relation to lower levels of cAMP. However, it is unclear whether it can be attributed to the effect on cyclic nucleotide binding affinity or the impact on downstream structural change and channel gating. We used isothermal titration calorimetry to determine the binding of cAMP to a naturally occurring tetramer of the C-linker and CNBD of the HCN4 channel. For both the mutant and wild type, we found that cAMP bound to the protein with negative cooperativity, and the high and low binding affinities for cAMP were not significantly different between WT and mutant. We also reported a crystal structure of the HCN4 C-terminus carrying the R524Q mutation. The mutation did not cause significant change to the global structure. However, it impacts a salt bridge with the transmembrane S4-S5 domain which plays an important role in keeping channel closed. Our results suggest the shift of the concentration-response relation in HCN4 R524Q mutant may be largely attributed to processes downstream that couple cAMP binding to channel opening.
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