Gating mechanism of hyperpolarization-activated HCN pacemaker channels

Abstract

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are essential for rhythmic activity in the heart and brain, and mutations in HCN channels are linked to heart arrhythmia and epilepsy. HCN channels belong to the family of voltage-gated K + (Kv) channels. However, why HCN channels are activated by hyperpolarization whereas Kv channels are activated by depolarization is not clear. Here we reverse the voltage dependence of HCN channels by mutating only two residues located at the interface between the voltage sensor and the pore domain such that the channels now open upon depolarization instead of hyperpolarization. Our data indicate that what determines whether HCN channels open by hyperpolarizations or depolarizations are small differences in the energies of the closed and open states, due to different interactions between the voltage sensor and the pore in the different channels.

Mapping Electromechanical Coupling Pathways in Voltage-Gated Ion Channels: Challenges and the Way Forward

“… 80 Furthermore, spHCN voltage dependence is completely inverted through the double mutation W355N (S4 C-terminus) and N370W (S5 N-terminus). 81 This critical role of S4-S5 interactions in closed-state stabilization resembles proposals for hERG gating, albeit with inverted voltage dependence.…”

Gating movements and ion permeation in HCN4 pacemaker channels

“…Thus, in the absence of lipids, the tight network of hydrophilic and hydrophobic interactions between S4 and S5 helices is lost, either partially (loss of hydrophilic interaction; Figure 4 D; Figure S13 B) or totally (loss of hydrophilic and hydrophobic interactions; Figure 4 E; Figure S13 C). In either case, these alterations include the disruption of the hydrogen bond between E403 (S4) and N421 (S5) ( Figures 4 D and 4E), which was previously shown by functional mutagenesis experiments to lead to channels with a large constitutive current component in HCN2 ( Chen et al., 2001a ) and the invertebrate Strongylocentrotus purpuratus spHCN ( Flynn and Zagotta, 2018 ; Ramentol et al., 2020 ). Second, the lower end of S5 tilts upward, pointing out of the planar array.…”

“…This time- and voltage-independent component makes up ∼5%–10% of the total conductance ( Proenza and Yellen, 2006 ; Proenza et al., 2002 ). The open structures may thus reflect an open state in the absence of voltage, further supported by the findings that (1) displacement of S5 in the lipid-free structures leads to disruption of the hydrogen bond between E403 and N421, which was shown to yield channels with a large constitutive current component in functional mutagenesis studies ( Chen et al., 2001a ; Ramentol et al., 2020 ); and (2) the displacement of S5 in the apo/LC structure is virtually identical to that seen in the structure of the constitutively open HCN1 mutant Y286D ( Chen et al., 2001a ; Lee and MacKinnon, 2019 ).…”

Electromechanical coupling mechanism for activation and inactivation of an HCN channel

“…HCN channels with a deletion of either the A′ helix or the S4–S5 linker maintain their hyperpolarization-dependent activation 14 , 32 , 33 , 50 . Instead, mutations of the S4 and S5 segments can eliminate the hyperpolarization-dependent activation and uncover depolarization-dependent activation 14 , 21 , 22 . This depolarization-dependent activation is eliminated by the addition of cAMP, reminiscent of the hyperpolarization-dependent inactivation in spHCN channels 14 .…”

“…The rearrangement of the VSD of HCN channels in response to voltage is generally similar to that in other depolarization-activated channels, with the positively-charged S4 helix moving downward upon hyperpolarization (upward upon depolarization) 15 – 20 . Interestingly, minimal mutations in the intracellular sides of the S4 and S5 helices could reverse the gating polarity of HCN channels, making them depolarization activated 14 , 21 , 22 . Some mutations produce channels that are both hyperpolarization activated and depolarization activated 14 , 22 , suggesting that the coupling pathways for hyperpolarization and depolarization activation are distinct.…”