Allostery modulates the beat rate of a cardiac pacemaker

The S672R mutation in heart cell ion channels leads to low heart rates and arrhythmia by an unknown route. A multifaceted NMR analysis now demonstrates that this mutant impacts allosteric coupling in domains inside of the cell to change channel activation, providing a mechanistic explanation for phenotypic outcomes.

The continuous and consistent beating of a heart is an amazing thing. Cells in the wall of the heart, in the sinoatrial node, spontaneously produce the electrical impulse that keeps the heart muscle moving. This current, in turn, depends on specialized hyperpolarization-activated cyclic nucleotide-gated (HCN) 2 ion channels that create ion influxes, making cells easier to activate (1). The importance of the HCN channels to this process is reflected by the discovery that the S672R mutation in the cAMP-binding domain (CBD; also known as the cyclic nucleotide-binding domain of HCN4, the most abundant channel isoform), is a direct cause of familial lower heart rate or arrhythmia disorder (2). Phenotypically, the mutation causes a negative shift in the HCN channel-activation voltage, making the channel more difficult to activate, as well as accelerating its deactivation (3). However, the mechanistic details underlying this phenotype have been less clear. A new NMR analysis of the S672R mutation from Boulton et al. (4) sheds light on these aspects, pointing to HCN channel dynamics as the critical factor in modulating the beat rate.
HCN channels integrate multiple signals to control ion flux across the membrane. Channel opening is primarily regulated by transmembrane elements that sense membrane hyperpolarization (5). However, the voltage for HCN activation is also regulated by tetramerization, mediated by an intracellular domain called the C-linker, which reduces the required voltage (6). Tetramerization can occur only when cAMP is bound, as this event induces a conformational change (Fig. 1A) in the other intracellular domain, the CBD, that would otherwise clash with the tetrameric C-linker conformation; in the apo form, then, auto-inhibition is observed (6). Surprisingly, the crystal structure of cAMP bound to an hHCN4 C-linker/CBD construct carrying the S672R mutation, determined by Xu et al. (7), revealed no substantial global conformational changes as compared with the wild-type structure except for a disordered loop on the cAMP-entry path. Together with the experimentally observed 10-and 3-fold decreases of cAMPbinding affinity via isothermal titration calorimetry and fluorescence anisotropy methods, respectively, Xu et al. (7) concluded that the S672R mutation simply weakened the interaction between cAMP and the channel, destabilizing the bound cAMP and promoting the closed state.
Boulton et al. (4) suspected that there may be more to the structural story than the static X-ray images conveyed. The authors used a suite of NMR techniques, including heteronuclear singlequantum coherence spectroscopy (NH-HSQC), a two-dimensional 1 H, 15 N-TROSY, along with subsequent chemical shift projection analysis (CHESPA), to examine the S672R-containing HCN4 (residues 563-724) in the apo and cAMP-bound holo form. Comparing their data to that previously collected for the wild-type protein (6) revealed an extensive perturbation of the dynamics in both apo and holo forms of the S672R mutant. First, they observed a constitutive shift toward the auto-inhibitory inactive conformation of S672R, which, in tandem with a simplified free-energy diagram, explains the negative shift in the activation voltage observed by electrophysiology (Fig. 1B). Second, they fitted k off values for the wild-type and S672R from HSQC analyses that indicated an ϳ6-fold faster k off value for the mutant, pointing to a S672Rinduced acceleration of cAMP release. This corresponding accelerated channel deactivation is consistent with the fact that patients carrying the S672R mutation show a significant drop in heart rate.
Several points make the Boulton et al. paper (4) significant. The manuscript is the first NMR investigation that transforms the dynamic profile of an HCN channel into a simple free-energy landscape for the wild type and the S672R variant in the cardiac pacemaker channels, illustrating clearly how allostery works through modulation of an auto-inhibitory mechanism and explaining precisely the corresponding observed phenotype. Of note, based on electron paramagnetic resonance (EPR) and NMR experiments, a recent study (8) indicated that different agonists (cAMP, cGMP, or cCMP) bound to the isolated CBD led to different degrees of conformational changes and extents of stabilization of the active conformation. However, corresponding electrophysiology experiments produced similar increases in the extents of channel activation for the three agonists. This implies that distinct conformational states of the isolated CBD might contribute equally to the release of autoinhibition for tetramerization of HCN and, in turn, ion channel activation.
In general, this study provides a striking example where NMR spectroscopy can help in explaining observations that cannot be fully explained by static crystal structures (7). Similar approaches can be adopted for other systems where the conformational changes caused by the allostery are not observed in X-ray structure due to crystal packing or other factors (9). A, the monomeric apo CBD NMR structure (Protein Data Bank code 2MNG, red ribbon) was superimposed with the tetrameric X-ray structure of cAMP-bound CBD (Protein Data Bank code 3OTF, green ribbon). The light-colored ribbons (primarily in the ␤-sheet) are the bases for superposition and the dark-colored ribbons reflect the conformational changes that originate from the cAMP-binding site, propagate through the C-helix into N3A (A-helix, EЈ-helix and FЈ-helix), and then facilitate C-linker tetramerization. The orientation of the Ser-672 side chain and cAMP (both shown in the space-fill model) reveals no direct interaction between them. B, in the free-energy landscape, conformational changes are depicted by a relative free-energy shift from the inactive apo state to the active cAMP-bound state. For the wild-type CBD, the relative free-energy description is given by two curves, one for the apo CBD (orange) with favorable "inactive" state and the other for cAMP-bound CBD (cyan) in favor of the "active" state, indicated by a relative free-energy difference, ⌬G WT. Boulton et al. (4) show the S672R mutant populates the inactive conformational state more than the active state, with the relative free-energy shift favorable for the inactive state and opposite to the active state (from wild-type to S672R mutant, indicated by the arrows). The free-energy difference of the S672R mutant in favor of the active bound state is given as ⌬G SR , shown as a green curve. The indication of ⌬G WT Ͼ ⌬G SR clearly reflects the more negative shift of activation voltage in S672R HCN4.