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LA JOLLA, Calif. & JUPITER, Fla.—A new study from Scripps Research published in the Proceedings of the National Academies of Sciences (PNAS) has solved the mystery of exactly how anesthetics can render patients unconscious.
According to Dr. Scott B. Hansen, molecular biologist and an associate professor of molecular medicine at Scripps, “The initiation of sleep involves a reversible loss of consciousness which appears very similar to anesthesia. Anesthesia is referred to as ‘putting someone to sleep.’ However, brainwaves of people sleeping haven’t correlated very well with anesthesia and [have] left science with more questions than answers.”
Using nanoscale microscopic techniques, plus experiments in living cells and fruit flies, Scripps scientists showed how clusters of lipids in the cell membrane serve as go-betweens in a two-part mechanism. Exposure to anesthesia causes the lipid clusters to move from an ordered state to a disordered one, and then back again—leading to subsequent effects that ultimately cause changes in consciousness.
“A molecular understanding affords a whole new opportunity to understand sleep and potentially find naturally occurring anesthetic-like molecules that may exist in the brain. These compounds are likely disrupting the ability of lapidated (e.g. palmitoylated) proteins to associate with cholesterol dependent lipid compartments,” says Hansen. “There are entire pathways to be discovered and targeted.”
This discovery by Hansen and chemist Dr. Richard Lerner settles a century-old scientific debate: Do anesthetics act directly on ion channels, or do they act on the membrane to signal cell changes?
“We think there is little doubt that this novel pathway is being used for other brain functions beyond consciousness, enabling us to now chip away at additional mysteries of the brain,” Lerner stated.
When asked if the breakthrough is applicable to anesthetics aside from ether, Hansen noted that “We showed chloroform, isoflurane, diethyl ether, xenon and propofol all have the same effect.”
“This is the granddaddy of medical mysteries. When I was in medical school at Stanford, this was the one problem I wanted to solve,” Lerner added. “Anesthesia was of such practical importance I couldn’t believe we didn’t know how all of these anesthetics could cause people to lose consciousness.”
“People always ask me ‘How can we still not understand how anesthesia work?’ Up until now I’ve kind of shrugged my shoulders and said, ‘it is as puzzling to me as it is to you.’ But now I have a glimmer of understanding why,” explains Hansen. “One [reason] was a lack of the right tools. The structures are small, you can’t see them without a super-resolution microscope. Those microscopes have only been commercially available the last six years or so.”
Using a microscope called dSTORM, which is short for “direct stochastical optical reconstruction microscopy,” researchers bathed cells in chloroform and watched something that looked like the opening break shot in a billiards game. Hansen said that exposing the cells to chloroform strongly increased the diameter and area of cell membrane lipid clusters called GM1. What he was looking at was a shift in the GM1 cluster’s organization from a tightly packed ball to a disrupted mess. As it grew disordered, GM1 spilled its contents, among which was an enzyme called phospholipase D2 (PLD2).
After tagging PLD2 with a fluorescent chemical, Hansen could watch as PLD2 moved away from GM1 and over to a different, less-preferred lipid cluster called PIP2. This activated key molecules within PIP2 clusters, including TREK1 potassium ion channels and their lipid activator, phosphatidic acid (PA). The activation of TREK1 freezes neurons’ ability to fire, and leads to loss of consciousness, Hansen explained.
“TREK-1 is an analgesic channel that attenuates pain. Our research suggests anesthetics are changing a threshold rather than blocking a pathway,” Hansen points out. “Hence, rather than binding to the channel and inhibiting it, the anesthetics changed the threshold of when a signaling molecule is released.”
“The mechanism in this study shows one way the body shifts a threshold for neuronal activity, and based on the role of TREK-1 in pain, we should be to regulate pain thresholds better,” he continues. “I suspect drugs modulating sleep, anxiety and other mood disorders will be similar. Understanding TREK-1 is like opening up the hood of a car and seeing the parts of a motor for the first time. The first look is a little complicated, but then a few basic concepts stand out.”
The researchers found that deleting PLD expression in Drosophila melanogaster flies rendered them resistant to the effects of sedation. The flies required double the exposure to the anesthetic to demonstrate the same response.
“All flies eventually lost consciousness, suggesting PLD helps set a threshold, but is not the only pathway controlling anesthetic sensitivity,” Lerner and Hansen wrote in their PNAS paper. The discoveries raise tantalizing new possibilities that could explain other mysteries, including the molecular events that lead to sleep.
“We think this is fundamental and foundational, but there is a lot more work that needs to be done, and it needs to be done by a lot of people,” stated Hansen.
Lerner agrees: “People will begin to study this for everything you can imagine: sleep, consciousness, all those related disorders. Ether was a gift that helps us understand the problem of consciousness. It has shined a light on a heretofore unrecognized pathway that the brain has clearly evolved to control higher-order functions.”
“I often think about the broad implications [of this research]. The lipid structures are fundamental to all cells, not just neurons, and cholesterol appears to be the main regulator,” adds Hansen. “The role of cholesterol in many metabolic diseases is very poorly understood, and no doubt a problem with understanding cholesterol-dependent lipid domains at the level we have shown here.”
“We have shown a particular example with TREK-1, but there are many channels involved in anesthesia, and each one will likely have small differences that need working out. Those differences will be opportunities for more refined modulation. Many channels like GABAA have the hallmarks of TREK-1 (palmitoylation and PIP2 binding),” he concludes. “It will likely take some time for other labs working in anesthesia to get the right microscope and design the correct experiments. We need to find all the players that are moving in and out of these lipid structures in response to anesthetics.”