Structural basis for modulation and agonist specificity of HCN pacemaker channels

Abstract

The family of hyperpolarization-activated, cyclic nucleotide-modulated (HCN) channels are crucial for a range of electrical signalling, including cardiac and neuronal pacemaker activity, setting resting membrane electrical properties and dendritic integration. These nonselective cation channels, underlying the I(f), I(h) and I(q) currents of heart and nerve cells, are activated by membrane hyperpolarization and modulated by the binding of cyclic nucleotides such as cAMP and cGMP. The cAMP-mediated enhancement of channel activity is largely responsible for the increase in heart rate caused by beta-adrenergic agonists. Here we have investigated the mechanism underlying this modulation by studying a carboxy-terminal fragment of HCN2 containing the cyclic nucleotide-binding domain (CNBD) and the C-linker region that connects the CNBD to the pore. X-ray crystallographic structures of this C-terminal fragment bound to cAMP or cGMP, together with equilibrium sedimentation analysis, identify a tetramerization domain and the mechanism for cyclic nucleotide specificity, and suggest a model for ligand-dependent channel modulation. On the basis of amino acid sequence similarity to HCN channels, the cyclic nucleotide-gated, and eag- and KAT1-related families of channels are probably related to HCN channels in structure and mechanism.

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

“…The first structural information about the C-linker (approximately 80 residues) that connects the CNBD with the transmembrane segment S6 was reported by crystallization of the HCN2 CNBD (Zagotta et al, 2003). In the crystal structure, the four C-linker subunits including the CNBD are assembled as tetramers with a four-fold axis of rotational symmetry, which is consistent with the tetrameric channel architecture.…”

“…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).…”

“…The C-linker is important in CNG and HCN channels, because it links the CNBDs to the ion channel gating machinery, and the majority of all intersubunit interactions of the four subunits in the crystal structure are provided between the C-linker sub units without interactions between the CNBDs. 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.…”

“…Here, the structure of the C-terminal fragment of the murine HCN2 channel was solved by X-ray crystallography and consisted of the CNBD and the C-linker region (mHCN2 CNBD) in the presence of cAMP or cGMP (Zagotta et al, 2003). The protein core of the mHCN2 CNBD is characterized by a β roll, consisting of eight antiparallel aligned β-strands, topped by a helical bundle of four α-helices (Figure 2).…”

“…A detailed structurefunction analysis of CNG and HCN channel CNBDs was initiated (see information given in Table S1) when the first CNBD crystal structures of the eukaryotic HCN2 and prokaryotic MloK1 channel were reported (Zagotta et al, 2003;Clayton et al, 2004).…”