Key result
The αDE helices downstream of the cyclic nucleotide binding domain act as an affinity switch, increasing cAMP binding affinity in HCN channels by approximately 30 to 40-fold.
Why the study?
It remained unclear why the isolated cyclic nucleotide binding domain of HCN channels displays ten to hundred times lower affinity for cAMP in vitro than the full-length channel in patch experiments.
The discovery of an affinity switch (αDE helices) in HCN channels explains the discrepancy in cAMP affinity between isolated domains and full-length channels, revealing a novel regulatory mechanism for pacemaker activity.
Resolves cAMP affinity discrepancy in HCN channels via switch; leaves open physiological role in pacemaking.
Binding of cAMP to Hyperpolarization activated cyclic nucleotide gated (HCN) channels facilitates pore opening and underlies beta adrenergic stimulation of heart rate. It is unclear why the isolated cyclic nucleotide binding domain (CNBD) displays in vitro ten to hundred times less affinity for cAMP than the full-length channel in patch experiments (micromolar vs nanomolar). Here we resolve the incongruity by showing that HCN channels are endowed with an affinity switch for cAMP, so far overlooked. Alfa helices D and E (αDE), downstream of the CNBD, form a helix-turn-helix motif that binds to and stabilizes the CNBD in a high affinity state. These helices were simply not included in previous experiments conducted with the isolated CNBD fragment of HCN2 and their addition increases the KD by about 40-fold in this fragment, while their deletion in patch clamp experiments, reduces the EC50 of HCN4 current by the same extent. The results further highlight that αDE helices interact with the same element of the CNBD, the alfa C helix (αC) that is the target of another controller of HCN cAMP affinity, the beta subunit TRIP8b. Our results uncover a mechanism whereby changes in binding affinity, rather than changes in cAMP concentration, can modulate HCN channels, adding another layer to the complex regulation of their activity.
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Porro et al. (2023) studied this question. Deletion of αDE helices vs. Full-length HCN channels was evaluated on cAMP binding affinity (KD). The αDE helices downstream of the cyclic nucleotide binding domain act as an affinity switch, increasing cAMP binding affinity in HCN channels by approximately 30 to 40-fold.
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