Specific interactions critical for the gating of HCN channels

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are named appropriately given their ability to open upon hyperpolarization and are modulated by cyclic nucleotides through their cyclic nucleotide binding domain. HCN channels contain six transmembrane domains with S1-S4 forming the voltage-sensing domain and S5-S6 forming the pore domain. HCN channels are permeable to both potassium and sodium ions, playing a key role in a wide range bodily processes such as maintaining heart rate to neuropathic pain signaling. One key difference within the HCN family is, unlike mammalian HCN channels, sea urchin HCN (spHCN) channels undergo a rapid inactivation in the absence of cAMP. This inactivation process remains unclear, it has been proposed that the C-linker plays a role where binding of cAMP prevents a rotation in the A’ helix that is suggested to lead to the inactivation of the channel. Another study has highlighted a single point mutation, F459L, is able to abolish spHCN inactivation. Here we investigated both the C-linker and F459 residue as well as other sites involved in HCN channel gating using voltage clamp fluorometry and inside-out patch clamp in spHCN channels expressed in Xenopus laevis oocytes. Our results highlight some key areas across the HCN channel that may play a role in stabilizing the gate and the inactivation phenomenon seen in spHCN.