Altered cyclic nucleotide binding and pore opening in a diseased human HCN4 channel

A growing number of nonsynonymous mutations in the human HCN4 channel gene, the major component of the funny channel of the sinoatrial node, are associated with disease but how they impact channel structure and function, and, thus, how they result in disease, is not clear for any of them. Here, we study the S672R mutation, in the cyclic nucleotide-binding domain of the channel, which has been associated with an inherited bradycardia in an Italian family. This may be the best studied of all known mutations, yet the underlying molecular and atomistic mechanisms remain unclear and controversial. We combine measurements of binding by isothermal titration calorimetry to a naturally occurring tetramer of the HCN4 C-terminal region with a mathematical model to show that weaker binding of cAMP to the mutant channel contributes to a lower level of facilitation of channel opening at submicromolar ligand concentrations but that, in general, facilitation occurs over a range that is similar between the mutant and wild-type because of enhanced opening of the mutant channel when liganded. We also show that the binding affinity for cGMP, which produces the same maximum facilitation of HCN4 opening as cAMP, is weaker in the mutant HCN4 channel but that, for both wild-type and mutant, high-affinity binding of cGMP occurs in a range of concentrations below 1 mu M. Thus, binding of cGMP to the HCN4 channel may be relevant normally in vivo and reduced binding of cGMP, as well as cAMP, to the mutant channel may contribute to the reduced resting heart rate observed in the affected family.

Automated summary

An increasing number of nonsynonymous mutations in the HCN4 channel gene are associated with disease, but the impact of these mutations on channel structure and function, and the molecular mechanisms underlying the disease, are not well understood. In this study, the S672R mutation in the cyclic nucleotide-binding domain of the channel was investigated. It was found that the mutant channel has weaker binding to cAMP and cGMP, which may contribute to the reduced resting heart rate observed in the affected family.