The Role Of Hcn Channel Helices D And E In The Modulation Of Camp Affinity

Hyperpolarization-activated cyclic-nucleotide regulated (HCN) channels are non-selective cation channels controlling key physiological functions such as cardiac pacemaking and repetitive neuronal firing. They are activated by voltage and modulated by binding of cAMP to their cytoplasmic C-terminal region named cyclic nucleotide binding domain (CNBD). Altered cAMP responses caused either by single point mutations in the protein or by non-physiological levels of the cyclic nucleotide in the cell lead to pathological conditions both in the heart and in the nervous system. The recently obtained cryo-EM structure of human HCN1 in the bound and unbound forms, confirms previous NMR studies on cAMP-induced conformational changes and reveals the presence of two additional helices at the C-terminus of the CNBD which fold in the presence of cAMP. We have previously shown, by ITC and patch clamp, that deletion of these helices (D and E) decreases the affinity for cAMP in the most studied HCN isoforms (HCN1, HCN2 and HCN4). Here we introduced single point mutations in HCN4 isoform, designed either to delete the single E helix or to selectively perturb the folding of the D helix, with the aim to dissect the role of the single D and E helices in this regulatory mechanism. Moreover, different truncations of the D helix were analyzed in order to clarify its role in the folding of the preceding C helix, known to be a key element in the transition of the CNBD from the unbound to the bound state. The patch clamp experiments were performed in whole cell configuration using a new all-in-one integrated amplifier, the ePatch.