Search

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels 1 are essential for pacemaking activity and neural signalling 2,3 . Drugs inhibiting HCN1 are promising candidates for management of neuropathic pain 4 and epileptic seizures 5 . The general anaesthetic propofol (2,6-di-iso-propylphenol) is a known HCN1 allosteric inhibitor 6 with unknown structural basis. Here, using single-particle cryo-electron microscopy and electrophysiology, we show that propofol inhibits HCN1 by binding to a mechanistic hotspot in a groove between the S5 and S6 transmembrane helices. We found that propofol restored voltage-dependent closing in two HCN1 epilepsy-associated polymorphisms that act by destabilizing the channel closed state: M305L, located in the propofol-binding site in S5, and D401H in S6 (refs.  7,8 ). To understand the mechanism of propofol inhibition and restoration of voltage-gating, we tracked voltage-sensor movement in spHCN channels and found that propofol inhibition is independent of voltage-sensor conformational changes. Mutations at the homologous methionine in spHCN and an adjacent conserved phenylalanine in S6 similarly destabilize closing without disrupting voltage-sensor movements, indicating that voltage-dependent closure requires this interface intact. We propose a model for voltage-dependent gating in which propofol stabilizes coupling between the voltage sensor and pore at this conserved methionine-phenylalanine interface in HCN channels. These findings unlock potential exploitation of this site to design specific drugs targeting HCN channelopathies., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Background and Purpose: In models of neuropathic pain, inhibition of HCN1 is anti-hyperalgesic. 2,6-di-iso-propyl phenol (propofol) and its non-anesthetic congener, 2,6-di-tert-butyl phenol, inhibit HCN1 channels by stabilizing closed state(s)., Experimental Approach: Using in vitro electrophysiology and kinetic modeling, we systematically explore the contribution of ligand architecture to alkylphenol-channel coupling., Key Results: When corrected for changes in hydrophobicity (and propensity for intra-membrane partitioning), the decrease in potency upon 1-position substitution (NCO∼OH >> SH >>> F) mirrors the ligands' H-bond acceptor (NCO > OH > SH >>> F) but not donor profile (OH > SH >>> NCO∼F). H-bond elimination (OH to F) corresponds to a ΔΔG of ∼4.5 kCal mol -1 loss of potency with little or no disruption of efficacy. Substitution of compact alkyl groups (iso-propyl, tert-butyl) with shorter (ethyl, methyl) or more extended (sec-butyl) adducts disrupts both potency and efficacy. Ring saturation (with the obligate loss of both planarity and π electrons) primarily disrupts efficacy., Conclusions and Implications: A hydrophobicity-independent decrement in potency at higher volumes suggests the alkylbenzene site has a volume of ≥800 Å 3 . Within this, a relatively static (with respect to ligand) H-bond donor contributes to initial binding with little involvement in generation of coupling energy. The influence of π electrons/ring planarity and alkyl adducts on efficacy reveals these aspects of the ligand present towards a face of the channel that undergoes structural changes during opening. The site's characteristics suggest it is "druggable"; introduction of other adducts on the ring may generate higher potency inverse agonists., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The ability of a diverse group of agents to produce general anesthesia has long been an area of intense speculation and investigation. Over the past century, we have seen a paradigm shift from proposing that the anesthetized state arises from nonspecific interaction of anesthetics with the lipid membrane to the recognition that the function of distinct, and identifiable, membrane-embedded proteins is dramatically altered in the presence of intravenous and inhaled agents. Among proteinaceous targets, metabotropic and ionotropic receptors garnered much of the attention over the last 30 years, and it is only relatively recently that voltage-gated ion channels have clearly and rigorously been shown to be important molecular targets. In this review, we will consider the experimental issues relevant to two important ion channel anesthetic targets, HCN and K 2P ., (© 2018 Elsevier Inc. All rights reserved.)

Background: The thalamus is thought to be crucially involved in the anesthetic state. Here, we investigated the effect of the inhaled anesthetic xenon on stimulus-evoked thalamocortical network activity and on excitability of thalamocortical neurons. Because hyperpolarization-activated, cyclic nucleotide-gated cation (HCN) channels are key regulators of neuronal excitability in the thalamus, the effect of xenon on HCN channels was examined., Methods: The effects of xenon on thalamocortical network activity were investigated in acutely prepared brain slices from adult wild-type and HCN2 knockout mice by means of voltage-sensitive dye imaging. The influence of xenon on single-cell excitability in brain slices was investigated using the whole-cell patch-clamp technique. Effects of xenon on HCN channels were verified in human embryonic kidney cells expressing HCN2 channels., Results: Xenon concentration-dependently diminished thalamocortical signal propagation. In neurons, xenon reduced HCN channel-mediated Ih current amplitude by 33.4 ± 12.2% (at -133 mV; n = 7; P = 0.041) and caused a left-shift in the voltage of half-maximum activation (V1/2) from -98.8 ± 1.6 to -108.0 ± 4.2 mV (n = 8; P = 0.035). Similar effects were seen in human embryonic kidney cells. The impairment of HCN channel function was negligible when intracellular cyclic adenosine monophosphate level was increased. Using HCN2 mice, we could demonstrate that xenon did neither attenuate in vitro thalamocortical signal propagation nor did it show sedating effects in vivo., Conclusions: Here, we clearly showed that xenon impairs HCN2 channel function, and this impairment is dependent on intracellular cyclic adenosine monophosphate levels. We provide evidence that this effect reduces thalamocortical signal propagation and probably contributes to the hypnotic properties of xenon.

Hyperpolarization-activated cyclic nucleotide-regulated HCN channels underlie the Na+-K+ permeable IH pacemaker current. As with other voltage-gated members of the 6-transmembrane KV channel superfamily, opening of HCN channels involves dilation of a helical bundle formed by the intracellular ends of S6 albeit this is promoted by inward, not outward, displacement of S4. Direct agonist binding to a ring of cyclic nucleotide-binding sites, one of which lies immediately distal to each S6 helix, imparts cAMP sensitivity to HCN channel opening. At depolarized potentials, HCN channels are further modulated by intracellular Mg2+ which blocks the open channel pore and blunts the inhibitory effect of outward K+ flux. Here, we show that cAMP binding to the gating ring enhances not only channel opening but also the kinetics of Mg2+ block. A combination of experimental and simulation studies demonstrates that agonist acceleration of block is mediated via acceleration of the blocking reaction itself rather than as a secondary consequence of the cAMP enhancement of channel opening. These results suggest that the activation status of the gating ring and the open state of the pore are not coupled in an obligate manner (as required by the often invoked Monod-Wyman-Changeux allosteric model) but couple more loosely (as envisioned in a modular model of protein activation). Importantly, the emergence of second messenger sensitivity of open channel rectification suggests that loose coupling may have an unexpected consequence: it may endow these erstwhile "slow" channels with an ability to exert voltage and ligand-modulated control over cellular excitability on the fastest of physiologically relevant time scales.