Structure and Rearrangements in the Carboxy-Terminal Region of SpIH Channels

Abstract

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) ion channels regulate the spontaneous firing activity and electrical excitability of many cardiac and neuronal cells. The modulation of HCN channel opening by the direct binding of cAMP underlies many physiological processes such as the autonomic regulation of the heart rate. Here we use a combination of X-ray crystallography and electrophysiology to study the allosteric mechanism for cAMP modulation of HCN channels. SpIH is an invertebrate HCN channel that is activated fully by cAMP, but only partially by cGMP. We exploited the partial agonist action of cGMP on SpIH to reveal the molecular mechanism for cGMP specificity of many cyclic nucleotide-regulated enzymes. Our results also elaborate a mechanism for the allosteric conformational change in the cyclic nucleotide-binding domain and a mechanism for partial agonist action. These mechanisms will likely extend to other cyclic nucleotide-regulated channels and enzymes as well.

Crystallization and preliminary X-ray crystallographic characterization of a cyclic nucleotide-binding homology domain from the mouse EAG potassium channel

“…A molecular-replacement solution (log-likelihood gain = 28, Z score = 5.1) was only obtained when an ensemble of six superimposed models created by the Phyre server was provided to Phaser. The six search homology models were created from CNB-domain structures with PDB codes 1q3e, 1vp6 (21% identity; Clayton et al, 2004), 2ptm (17% identity; Flynn et al, 2007), 1cx4 (13% identity; Diller et al, 2001), 1ne6 (18% identity; Wu et al, 2004) and 2pqq (18% identity; Midwest Center for Structural Genomics, unpublished work). Electron-density maps calculated after partial refinement of the molecular-replacement solution in PHENIX (Adams et al, 2010) showed features that were not present in the search models, confirming the validity of the solution.…”

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

“…Interestingly, in cGMP-bound structures the cGMP molecule exists in the syn configuration, thereby facilitating a hydrogen-bond interaction of the purine ring N2 atom with residue T592 located in the β roll (Flynn et al, 2007). Several residues in helix αC (R632, R635, I636 and K638) are reported to increase selectivity of HCN2 channels for cAMP rather than for cGMP (Flynn et al, 2007;Zhou and Siegelbaum, 2007), however the reason for this selectivity is poorly understood.…”

“…Several residues in helix αC (R632, R635, I636 and K638) are reported to increase selectivity of HCN2 channels for cAMP rather than for cGMP (Flynn et al, 2007;Zhou and Siegelbaum, 2007), however the reason for this selectivity is poorly understood.…”

“…So far, no structural information is available for the C-linker in CNG channels. However, the C-linker and CNBD structure from an invertebrate HCN channel designated SpIH, which shares sequence homology with CNG and HCN channels (Figure 4), shows a similar fold to that of HCN channels, suggesting that the C-linker of CNG channels exhibits a similar fold (Flynn et al, 2007). 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.…”