Abstract
Kim ED, Wu X, Lee S, Tibbs GR, Cunningham KP, Di Zanni E, Perez ME, Goldstein PA, Accardi A, Larsson HP, Nimigean CM. Nature. 2024;632(8024):451–459. PMID: 39085604. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels1 are essential for pacemaking activity and neural signalling.2,3 Drugs inhibiting HCN1 are promising candidates for management of neuropathic pain4 and epileptic seizures.5 The general anaesthetic propofol (2,6-di-iso-propylphenol) is a known HCN1 allosteric inhibitor6 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.
Commentary
If you’ve ever undergone surgery, you’ve likely benefited from propofol, a fast-acting intravenous anesthetic widely used for its ability to induce and maintain sedation with remarkable precision. Known for its rapid onset and quick recovery time, propofol exhibits both analgesic and hypnotic (sleep-inducing) effects, making it a preferred choice in clinical settings. Propofol gained widespread media attention after being implicated in the death of pop icon Michael Jackson, who succumbed to fatal respiratory depression after misusing the drug as a sleep aid without appropriate medical supervision.
Propofol's analgesic effects are linked to its action on hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, specifically HCN1. 1 Despite its clinical importance, the structural basis for propofol's interaction with HCN channels remained unclear until recently. In a groundbreaking study, Kim et al., 2 from the labs of Peter Larsson and Crina Nimigean, uncovered the molecular mechanism of propofol's action on HCN channels and its potential to restore function in epilepsy-related HCN1 mutations. These findings not only deepen our understanding of propofol but also suggest new avenues for treating HCN-related epilepsy.
HCN channels, comprising four isoforms (HCN1–4), are crucial for generating rhythmic electrical activity in brain and heart pacemaker cells, where they mediate Ih and If (“funny”) currents, respectively. 3 Mutations in HCN1–3 are associated with epilepsy, while HCN4 mutations disrupt cardiac rhythm due to impaired sinoatrial node function. 3
Structurally, HCN channels belong to the voltage-gated K+ (Kv) channel superfamily. 4 Similar to Kv channels, HCN channels form tetramers, with each subunit containing six transmembrane segments (S1–S6). The S1–S4 segments create the voltage-sensing domain (VSD), while S5–S6 form the pore domain. Unlike Kv channels, which activate upon depolarization to allow K+ flux, HCN channels are activated by hyperpolarization, permitting the flux of K+ and Na+ ions.
Differences in electromechanical coupling between the VSD and pore domain underlie the distinct gating mechanisms of HCN and Kv channels. 5 Kv channels primarily use a canonical mechanism involving a long S4–S5 linker interacting with the S6 segment. They also engage in non-canonical coupling via non-covalent interactions between S4, S5, and S6. HCN channels, with a shorter S4–S5 linker, are believed to rely on this non-canonical pathway, though the precise molecular details remain unclear.
Propofol preferentially inhibits HCN1 channels by shifting their voltage dependence toward more hyperpolarized potentials. 6 Acting as an allosteric inhibitor, it binds outside the pore rather than blocking it directly. Kim et al. identified this elusive binding site using cryo-electron microscopy (cryo-EM), revealing that propofol binds in a groove between the S5 and S6 helices of adjacent subunits when the channel is closed. Molecular dynamics simulations and docking studies supported this finding.
Mutagenesis experiments further validated the S5/S6 groove as the binding site. Specifically, mutations M305E (on S5) and T384F (on S6) reduced propofol sensitivity, confirming the significance of this region. Kim et al. also examined propofol's effects on two epilepsy-associated HCN1 mutations, M305L (S5) and D401H (S6), which destabilize the closed state, resulting in leaky, voltage-insensitive channels. These mutations cause early infantile epileptic encephalopathy, a severe epilepsy marked by drug-resistant seizures and intellectual disability. 4 Remarkably, propofol restored proper voltage-dependent gating in both mutants.
To explore how propofol rescues these mutant channels, Kim et al. used cryo-EM to examine M305L mutants and found no structural defects in the VSD. Voltage-clamp fluorometry in spHCN channels (sea urchin homologs of HCN1) with M305-equivalent mutations confirmed that voltage sensor movements were intact despite the leak currents, implicating disrupted electromechanical coupling between the voltage sensor and pore gate. Additional mutations in residues homologous to F389 (S6), which contacts M305 (S5) in the closed state to form the propofol binding pocket, also exhibited voltage-independent currents despite normal voltage sensor movements. Finally, when propofol was applied to wild-type spHCN channels, it inhibited channel activity without affecting voltage-sensor dynamics.
In the model proposed by Kim et al., propofol stabilizes the closed state of HCN1 by binding to a mechanistic hotspot at the interface of M305 (S5) and F389 (S6) that contributes to non-canonical electromechanical gating. This interaction creates steric hindrance that prevents the S5/S6 helices from rotating to open the gate. In epilepsy-linked mutants, this interface is disrupted, causing voltage-independent leak currents due to decoupling the voltage sensor from the gate. Propofol effectively “glues” the voltage sensor and pore gate back together, restoring normal function.
Recently, however, the story grew more complex when another research group subsequently discovered a second propofol binding site using photoaffinity labeling and mass spectrometry. 7 In this latest study, Burtscher et al. 7 identified a different propofol binding pocket near Y234 in the S3 and S4 helices of the VSD. The reason Kim et al. did not detect the S3/S4 site is unclear. However, Burtscher et al. 7 speculated that it could be due to poor resolution in the S3–S4 loop in the cryo-EM structures or the cryo-EM freezing process, which may have extracted propofol from the S3/S4 site. Together, these studies suggest propofol binds to at least two distinct sites: the S3/S4 VSD and the S5/S6 pore domain. The identification of multiple propofol binding sites underscores the complexity of these channels and the need for continued research to understand how HCN channels are modulated.
Although propofol's ability to rescue HCN1 channel epilepsy mutants is promising, its anesthetic properties limit its practical use as an epilepsy treatment. Propofol also activates GABAA receptors, potentially enhancing seizure control but causing dangerous sedative effects and respiratory depression, 8 as tragically demonstrated in Michael Jackson's death. This is especially concerning in epilepsy, where respiratory suppression increases the risk of sudden, unexpected death in epilepsy (SUDEP). Additionally, propofol inhibits cardiac HCN4 channels, 3 increasing the risk of bradycardia and SUDEP.
Despite these limitations, propofol's allosteric mechanism offers exciting opportunities for developing novel and more effective epilepsy therapies via rational drug design. Currently, the only Food and Drug Administration-approved drug for HCN channels is ivabradine, which is used to treat heart failure by blocking the open pore of cardiac HCN4 channels. 9 A downside of pore-blocking drugs is that they often lack specificity and have a higher risk of adverse effects. 10 In contrast, allosteric modulators bind to non-conserved sites, offering the potential for greater specificity and reduced off-target toxicity. 10 They also enable more precise fine-tuned control of channel activity by modulating function indirectly at sites spatially discrete from the pore or where ligands bind. 10 Exploiting propofol's allosteric action could lead to new analogs with superior HCN1 selectivity, without affecting HCN4 channels or GABAA receptors, avoiding cardiac and sedative side effects. Given the high drug resistance in HCN1-related epilepsy, these targeted therapies could provide much-needed precision treatment for affected patients.
