Energetic and structural impact of cyclic nucleotide binding to hyperpolarization-activated cyclic nucleotide-gated channels

Hyperpolarization-activated Cyclic Nucleotide-gated, HCN, channels contribute to the membrane potential of excitable cells including pacemaker cells of the heart and neurons in the brain. By binding to the inner side of the HCN channel, cAMP facilitates channel opening, but the underlying mechanism has been mainly inferred from relating cAMP concentration to the degree of facilitation. Concentration-response relations reflect the tightly coupled process of cAMP binding and channel opening. The strength of binding and how it is linked to channel opening is not known. Furthermore, cAMP facilitation is not equal among the four mammalian HCN isoforms and the extent to which cAMP binding affinity contributes to these differences is not known.My experiments support the conclusion that cAMP binds to one site of the isolated tetrameric C-terminus of HCN2 and HCN4 with high affinity and to three sites with low affinity revealing negative cooperativity. In contrast, only low affinity binding was observed in HCN1 with energetics of binding that were similar to those of the low affinity binding to HCN2. Cyclic AMP enhanced oligomerization of the HCN2 C-terminus in solution, but had a negligible effect on oligomerization of the HCN1 C-terminus. Oligomerization in solution is thought to reflect the formation of a gating ring in the intact channel that facilitates opening. Together, this suggests that HCN1 functions as though already disinhibited, explaining its easier opening in the absence of cAMP, its smaller facilitation of opening, and lack of negative cooperativity upon cAMP binding. Lysine substitution at residue 488 of HCN2, initially identified in an individual with idiopathic generalized epilepsy, eliminated negative cooperativity and reduced oligomerization of the isolated C-terminus upon cAMP binding. This likely reflects a decrease in its ability to form a gating ring in the intact channel and explains the reported inhibition of opening by this mutation.The work presented in this thesis demonstrates the value of studying the C-terminus of the HCN channel in isolation to uncover the mechanism by which the HCN C-terminus and cAMP binding control channel opening that would otherwise be hidden by functional experiments.Medicine, Faculty ofCellular and Physiological Sciences, Department ofGraduat