HCN DOMAIN: A KEY REGULATOR OF HCN CHANNEL GATING

Hyperpolarization-activated and cyclic nucleotide–gated (HCN1-4) channels control key physiological functions such as cardiac pacemaking and repetitive neuronal firing. Their molecular architecture comprises a voltage sensor and a pore domain that are membrane-embedded and a cytosolic soluble part that includes the cyclic nucleotide binding domain (CNBD) connected to the pore via a C-linker. These channels are primarily activated by hyperpolarizing membrane voltage that opens the pore allowing the flux of a slow time-dependent depolarizing inward current known as If and Ih in the heart and in the brain, respectively. HCN channels can be further regulated by the direct binding of cyclic nucleotides to the CNBD that enhances channel opening by shifting the relationship between opening and membrane hyperpolarization to more positive potentials. Current knowledge of their mechanism of gating includes a downward movement/rotation of the voltage sensor that allosterically increases the affinity for cAMP in the CNBD, suggesting a direct interaction between the voltage sensor domain and the CNBD. The recently published single particle cryo-EM structure of HCN1 channel, constitutes the basis for this project that aims at understanding the cAMP-induced conformational changes leading to channel modulation. These are important questions to address because aberrant gating can lead to heart failures or epileptic seizures. Interestingly, the high resolution HCN1 structure shows that the N-terminus forms a 3 alfa-helical domain, called HCN domain. In the tetrameric assembly of the full length channel, the HCN domain of one subunit is inserted between the voltage sensor and the C-linker of the adjacent subunit. This suggests that the HCN domain could be the element coupling the cyclic nucleotide regulation whit the voltage sensitivity. By a combination of rational mutagenesis and functional analysis of the ion currents by patch-clamp, we found that the newly discovered HCN domain plays a crucial role in the modulation of the voltage dependence of the channel as well as in the cAMP response. Moreover, we show that by interacting with the CNBD in HCN2 but not in HCN1, the HCN domain regulates the affinity of the channel for the ligand. On the basis of these results we have prepared a synthetic HCN domain peptide that, when added to the channel, mimics the effect of cAMP. These promising results contribute not only to a better understating of the gating mechanism of the channel but also lay the basis for the design of drug molecules able to modulate HCN-mediated currents both in nervous system and heart.