Free energy landscape remodeling of the cardiac pacemaker channel explains the molecular basis of familial sinus bradycardia

Abstract

Edited by Norma AllewellThe hyperpolarization-activated and cyclic nucleotide-modulated ion channel (HCN) drives the pacemaker activity in the heart, and its malfunction can result in heart disorders. One such disorder, familial sinus bradycardia, is caused by the S672R mutation in HCN, whose electrophysiological phenotypes include a negative shift in the channel activation voltage and an accelerated HCN deactivation. The outcomes of these changes are abnormally low resting heart rates. However, the molecular mechanism underlying these electrophysiological changes is currently not fully understood. Crystallographic investigations indicate that the S672R mutation causes limited changes in the structure of the HCN intracellular gating tetramer, but its effects on protein dynamics are unknown. Here, we utilize comparative S672R versus WT NMR analyses to show that the S672R mutation results in extensive perturbations of the dynamics in both apo-and holo-forms of the HCN4 isoform, reflecting how S672R remodels the free energy landscape for the modulation of HCN4 by cAMP, i.e. the primary cyclic nucleotide modulator of HCN channels. We show that the S672R mutation results in a constitutive shift of the dynamic auto-inhibitory equilibrium toward inactive states of HCN4 and broadens the free-energy well of the apo-form, enhancing the millisecond to microsecond dynamics of the holo-form at sites critical for gating cAMP binding. These S672R-induced variations in dynamics provide a molecular basis for the electrophysiological phenotypes of this mutation and demonstrate that the pathogenic effects of the S672R mutation can be rationalized primarily in terms of modulations of protein dynamics.

Mechanism of allosteric inhibition in the Plasmodium falciparum cGMP-dependent protein kinase

“…3 suggest that chemical shifts are ideally suited to investigate the dynamical allosteric transitions underlying PfPKG inhibition. This notion is fully consistent with the fast exchange between the inactive and the active states of CBDs (27)(28)(29)(30)(31)(32)(33), whereby the observed NMR peak positions are population-weighted averages of the pure inactive and active ppm values. Hence, chemical shifts report on the position of the dynamic inactive-active CBD conformational equilibrium.…”

“…Hence, chemical shifts report on the position of the dynamic inactive-active CBD conformational equilibrium. The CHemical Shift Projection Analysis (CHESPA) is an effective means to evaluate the perturbation of such dynamic equilibria caused by ligand modifications (28,29,31,32). In the CHESPA analysis, the NMR peak positions of the cGMP-analog bound sample are evaluated relative to a reference vector connecting the peaks of the apo inactive and the cGMP-bound active samples (Fig.…”

The structure of the apo cAMP‐binding domain of HCN4 – a stepping stone toward understanding the cAMP‐dependent modulation of the hyperpolarization‐activated cyclic‐nucleotide‐gated ion channels

“…In fact, allosteric modulators are known to elicit a more selective control of function compared to inhibitors targeting orthosteric sites, which are often conserved among multiple homologous targets within the same protein family. Drugs targeting allosteric sites are likely to exhibit higher selectivity, given the lesser evolutionary pressure for conservation at loci remote from the active sites . The structure of the auto‐inhibited apo HCN4 CBD provides a first step towards developing allosteric HCN modulators.…”

Allostery modulates the beat rate of a cardiac pacemaker

“…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.…”

“…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.…”