Tetramerization Dynamics of C-terminal Domain Underlies Isoform-specific cAMP Gating in Hyperpolarization-activated Cyclic Nucleotide-gated Channels

Abstract

Background: HCN2 and HCN4 respond to cAMP, whereas HCN1 does not. Results: The C-linker plus CNBD of HCN2 and HCN4 show cAMP-induced tetramerization, whereas that of HCN1 contains prebound cAMP and is tetrameric. Conclusion: HCN1 does not respond to the addition of cAMP because its CNBD contains cAMP already. Significance: Tetramerization of the C terminus controls ligand gating in HCN channels.

Structural snapshot of cyclic nucleotide binding domains from cyclic nucleotide-sensitive ion channels

“…This would explain why HCN1 shows a weaker response to cAMP in contrast to the HCN2 and HCN4 isoforms, because the HCN1 CNBD is already partly present in a tetrameric state under non-saturating ligand conditions. This observation was confirmed by voltage clamp experiments, which showed that a shifted position to a more positive value of the half-activation voltage (V 1/2 ) in HCN1 correlates with its behavior to be present as a tetramer in vitro (Lolicato et al, 2011). Lolicato et al solved the crystal structures of the C-linker region and CNBD from the mouse HCN1, human HCN2, and mouse HCN4 channel all in the presence of cAMP.…”

“…To address this point, Xu et al solved the crystal structure of the cAMP-bound human HCN4 CNBD, including the C-linker (hHCN4 CNBD) (Xu et al, 2010). The overall structure of the hHCN4 CNBD and subsequently the mouse HCN4 CNBD (Lolicato et al, 2011) are very similar to the previously determined mHCN2 CNBD structure (Figure 2) and showed a four-fold symmetry arrangement of the protein with intersubunit interactions predominantly formed by the C-linkers from neighboring subunits. Functional characterization through electrophysiological experiments of a chimeric HCN channel consisting of the hHCN4 CNBD and C-linker connected to the mHCN2 transmembrane part (Figure 4) showed a three-fold reduction in response to cAMP when compared to the full-length mHCN2 channel (Xu et al, 2010).…”

“…tetrameric assembly of these domains as shown by equilibrium sedimentation experiments of the CNBD with the C-linker (Zagotta et al, 2003;Lolicato et al, 2011). Thus, the C-linkers appear to play a crucial role in allosteric modulation of channel gating and show high sequence similarity among each other ( Figure 4).…”

“…Surprisingly, cAMP dose-response curves for HCN1 and HCN2 obtained from inside-out patch clamp recordings showed similar nanomolar affinities for the activation of both channels, suggesting that the minor shift of the HCN1 activation curve is not related to a lower binding affinity of the HCN1 isoform for cAMP Wang et al, 2001). Interestingly, a combined approach of structural, biochemical and biophysical characterization of the HCN1, HCN2, and HCN4 CNBDs including the C-linker regions revealed that the HCN1 construct tetramerizes when half-saturated with cAMP (Lolicato et al, 2011), and this observation contrasts to the HCN2 and HCN4 isoforms that require saturating amounts of cAMP (Zagotta et al, 2003;Xu et al, 2010). This would explain why HCN1 shows a weaker response to cAMP in contrast to the HCN2 and HCN4 isoforms, because the HCN1 CNBD is already partly present in a tetrameric state under non-saturating ligand conditions.…”

“…The C-linker is composed of six α-helices (αA′ to αF′) and the first two helices of each subunit (αA′ and αB′) form an antiparallel 'helix-turnhelix' motif that interacts with helix αC′ and αD′ of the neighboring subunit, i.e., like an 'elbow' resting on the 'shoulder' of its neighbor (Zagotta et al, 2003; Figure 5A). This conformation, designated as the 'gating ring' (Johnson and Zagotta, 2005), was first presented for HCN2 and later by structures of different HCN isoforms (Zagotta et al, 2003;Flynn et al, 2007;Craven et al, 2008;Taraska et al, 2009;Lolicato et al, 2011;Xu et al, 2010Xu et al, , 2012.…”