By David N. Leff

When surgeon John C. Warren excised a tumor from the neck of 20-year-old Gilbert Abbott, at Massachusetts General Hospital, on October 16, 1846, his young patient felt no pain. Warren's colleague, dentist William Morton, had put the lad to sleep by having him inhale from a sponge soaked in sulfuric ether.

That operation, one and one-half centuries ago, was the first surgical use of a general anesthetic.

No one at the time had the slightest idea why ether, a clear, volatile, inflammable, explosive liquid, could cause a person to lose consciousness, and wake up none the worse. In fact, from that day to this, the mechanism by which anesthetics act on the central nervous system has remained something of a black box.

An article in today's Nature lifts one corner of the box's lid another crack. Its title: "Sites of alcohol and volatile anaesthetic action on GABA_A and glycine receptors." Its first author is molecular pharmacologist S. John Mihic, at Wake Forest University's Bowman Gray School of Medicine, in Winston-Salem, N.C. The paper's two senior co-authors are Neil Harrison, University of Chicago, and R. Adron Harris, University of Colorado Health Sciences Center, Denver.

"We concluded," Mihic told BioWorld Today, "that alcohol and volatile anesthetics have discrete binding sites on GABA (gamma-aminobutyric acid) and glycine receptors. These sites potentiate the function of those receptors by the anesthetic substances." He continued, "We identified a molecular site that is important for that mechanism, which nobody in the past has ever done."

Previous Theory Missed The Mark

In the past, for many years, Mihic went on, "people thought that anesthetics and alcohols had non-specific sites of action. That they fluidized the lipid content of cell membranes, and so disordered them.

He recalled the long-standing Meyer-Overton theory of narcosis, propounded by two researchers at the turn of the century, that the lipid solubility of a compound correlated directly with its anesthetic potency.

"This went on for most of our century," Mihic said. "Then, about 15 years ago, a couple of guys in England, Nicholas Franks and William Lieb, started arguing that no, perhaps these compounds, instead, are acting at much more specific sites on proteins rather than on lipids. They showed this on anesthetics and alcohols in a lipid-free model system. It demonstrated that these compounds could act at pharmacologically active concentrations on proteins that had no lipid surrounding them."

More recently, while a post-doctoral student at the University of Colorado, Mihic recalled, "Harris and I showed that some very lipid-soluble compounds, in stark contrast to what Overton and Meyer would have predicted, are non-anesthetic. And these compounds, which do not produce anesthesia in vivo, also have no effects on GABA or glycine receptors." Essentially, glycine acts to inhibit neurons in the spinal cord; GABA inhibits them in the brain.

Those breaks with orthodoxy led Mihic and his colleagues "to consider more strongly that these protein sites of action were actually important to the mechanism of anesthesia."

That time in the mid-1990s was ripe for an alternative theory as to how anesthetics render people unconscious, and how alcohol intoxicates them.

"We started thinking," Mihic recounted, "that those compounds could be acting on chloride ion channels. It's a very reasonable assumption, because ion channels rapidly alter synapse activity."

GABA and glycine receptor molecules each consist of five molecular subunits arranged like the slices of a pie. Each makes a part of the ion channel, which is in the center. The subunits assemble to form a functional receptor.

Mihic and his Nature co-authors constructed receptors with all five of their sites occupied by the same subunit and mix-matched them to produce chimeric molecules. "We thought," he explained, "that these chimeras might give us an idea of exactly where the molecular sites are on these anesthetic and ethanol-sensitive receptors that exert the potentiating effects on the cell membranes. So we just started mutating our way through their transmembrane domains."

Then, when they mutated the glycine receptor to replace a serine amino acid with an isoleucine, "The resulting glycine receptor was completely insensitive to the potentiating effects of ethanol, but still sensitive to enflurane."

Mihic made the point that "this was done on proteins that are widely believed to be important for the actions of alcohol and volatile anesthetics, not only in lab animals, but in humans."

Might such a finding find utility, say, in treating alcoholism in humans?

"That's the next thing we want to go after," Mihic replied. "We're not sure of exactly how important the ethanol effects are on this particular chloride ion channel, on the GABA and glycine receptors. We're actually branching out into other related receptors to see if they have common sites of alcohol action."

Who Needs Better Anesthetics?

On the one hand, Mihic foresees that in the long term this research can "lead to the design of better anesthetics." But on the other hand, he pointed out, "I think current general volatile anesthetics are pretty good. They don't have major side effects, and it's my impression that the drug companies figure it may never be cost-effective to replace them."

He regards as the most promising next step "using modern molecular biology techniques to construct transgenic animals, engineered with receptors insensitive to alcohol or anesthetics. Then," he pointed out, "one could ask: 'When I give the mouse ethanol, is it more sensitive or less sensitive? Is the animal more or less prone to drink alcohol?' Those transgenics would allow us to understand which particular receptors are important for specific aspects of anesthesia or alcohol use."

This project, Mihic said, "is still in the design stage. We're nowhere close. What we want to do before embarking on a project like this is produce an animal in which the glycine or GABA receptor is as normal as possible, except for being selectively insensitive to ethanol or anesthetics. And none of us has any hands-on experience in creating transgenic animals, so we would have to collaborate with other people to get this going." *