Epilepsy is a condition that occurs when the brain’s electrical signaling misfires and the body experiences a seizure (1). Both hereditary and environmental factors contribute to epilepsy. Even though one in 26 people will develop epilepsy at some point during their lives, only a few Food and Drug Administration (FDA)-approved epilepsy medications exist. In a recent study, researchers discovered that propofol, a drug typically used for anesthesia, could treat certain kinds of epilepsy by targeting ion channels that become dysfunctional due to the disease (2).

Specialized channels scattered throughout the nervous and cardiovascular systems regulate the voltage signaling needed by neurons to fire and muscles to contract. Some of these channels, called hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, can become mutated, which results in the overactivation of neuronal firing. Mutations in HCN genes have been increasingly associated with some forms of epilepsy (3). Researchers have shown that propofol can inhibit HCN channels, specifically HCN1, but it was unclear how (4). That is where Weill Cornell Medicine biophysicist Crina Nimigean and her team come in.

“We've been working on ion channels and understanding how ion channels work at the molecular level for quite a while now,” said Nimigean, a coauthor on the new study. “So, this was not coming out of the blue. The out-of-the-blue part was the propofol.”

Nimigean and her team’s first goal was to understand where propofol’s inhibitory effect on HCN1 takes place. If propofol is the key and HCN1 is the lock, gaining insight into how the two fit together could help researchers pinpoint areas of interaction between them.

“We thought it would be a nice challenge to do this particular structural project because propofol is very small and difficult to detect in the complex [crystal] structure,” said Nimigean.

After many attempts, the team successfully purified the HCN1 lock with its propofol key using cryo-electron microscopy. They found that propofol inactivates HCN1 by binding in the core of the protein, the nexus of signaling activity.

To validate the propofol binding site, the team mutated one of the HCN1 amino acids with which the drug interacts: methionine at site 305. Normally, propofol would bind to the wildtype HCN1 channel and cause a complete loss of electrical signaling. However, after mutating M305 to a leucine, in the absence of propofol, the channel completely changed behavior. It was no longer able to regulate its voltage. After digging through the literature, the team uncovered that this mutation is connected to infantile epileptic encephalopathy (5).

“When we added propofol to this mutated HCN1, we saw that the voltage dependence was regained,” said Nimigean. “That had us scratching our heads for a while. What's going on here?”

To figure out what propofol does, the team needed to figure out how exactly the M305 mutation broke the channel in the first place. Coauthor Peter Larsson and his team at Linköping University mutated many residues in the binding pocket and found that methionine and phenylalanine must interact for signaling to occur. 

“The effect of propofol binding on the wild type [protein] is that it strengthens this interface, forcing the HCN1 channel to remain closed,” said Nimigean. “While with the mutated M305L, which was now devoid of this coupling, propofol is like a glue that restores the interaction.”

When we added propofol to this mutated HCN1, we saw that the voltage dependence was regained. … That had us scratching our heads for a while. What's going on here? 
- Crina Nimigean, Weill Cornell Medicine

Nazzareno Avanzo, a biophysicist at the University of Montreal and an ion channel expert who was not involved in this study, said, “This is a really big step, and it solved this problem that we didn't know could be solved this way.” He added, “What was interesting about this paper for me was not only did they identify the potential binding site for an anesthetic drug that is known to interact with these channels, but they determined that this drug actually could rescue disease mutations, which I hadn't heard about before.”

This work opens the possibility of using propofol for the treatment of HCN1-related epilepsy and for identifying additional molecules that don’t have propofol’s drowsy side effects. 

“Propofol is a nice molecule, in terms of helping people, because it is already FDA-approved,” said Nimigean.

Avanzo added, “This has the potential to lead to the development of more specific treatments for patients with these epileptic mutations because now we're in the era of structural biology.”

References

1. Sirven, J.I. Epilepsy: A Spectrum Disorder. Cold Spring Harb Perspect Med  5, (2015).
2. Kim, E.D. et al. Propofol rescues voltage-dependent gating of HCN1 channel epilepsy mutants. Nature  632, 451–459 (2024). 
3. DiFrancesco, J.C. & DiFrancesco, D. Dysfunctional HCN ion channels in neurological diseases. Front Cell Neurosci  9, (2015).
4. Lyashchenko, A.K. et al. Propofol Inhibits HCN1 Pacemaker Channels by Selective Association with the Closed States of the Membrane Embedded Channel Core. J Physiol  583, 37–56 (2007).
5. Marini, C. et al. HCN1 mutation spectrum: from neonatal epileptic encephalopathy to benign generalized epilepsy and beyond. Brain  141, 3160–3178 (2018).