Nicotine is responsible for more than four million smoking-related deaths each year. Yet people still smoke. Why? One reason is the stranglehold of addiction, started when nicotine enhances the release of a neurotransmitter called dopamine, a chemical messenger that induces a feeling of pleasure. That's what smoking, presumably, is all about.

Knowing specifically which receptor molecules are activated by nicotine in the dopamine-releasing cells would be a promising first step in developing a therapeutic drug to help people kick the habit. But that's a challenging goal, since there are many cell receptor proteins, each in turn comprising a set of "subunit proteins" that may respond to nicotine, or instead, to a completely different chemical signal.

Going After Alpha4

California Institute of Technology postdoctoral scholar Andrew Tapper, Professor Allan Collins of the University of Colorado, seven other colleagues, and Henry A. Lester, the Bren Professor of Biology at Caltech, have determined that when receptors with a specific subunit known as alpha4 are activated by nicotine, it's sufficient for some addiction-related events, such as pleasure response, sensitization, and tolerance to repeated doses of nicotine. This research suggests that alpha4, and the molecules that are triggered in turn by alpha4, may prove to be useful targets for addiction therapies.

When cells communicate in the brain, nerve impulses jump chemically across a gap between two nerve cells called the synapse, using a neurotransmitter such as acetylcholine. Acetylcholine activates specific receptors on the post-synaptic nerve cell. This starts the firing of electrical impulses and, in cells that manufacture dopamine, that pleasure-inducing messenger is released as well. Having completed its task, acetylcholine is then rapidly broken down by an enzyme called acetylcholinesterase.

Nicotine Gene

It's a clever and wondrous biological machine, says Lester. But, he says, "nicotine is clever too, because it mimics acetylcholine." Worse, nicotine is not broken down by acetylcholinesterase. "So it persists at the synapse for minutes rather than milliseconds, and excites the post-synaptic neurons to fire rapidly for long periods, releasing large amounts of dopamine. Most scientists believe that's a key reason why nicotine is so addictive."

Previous work in several laboratories in the 1990s had suggested that, of the many so-called nicotinic acetylcholine receptors, one consisting of subunits called alpha4 and beta2 was important for nicotine addiction. This had been determined by the use of so-called "knockout" mice.

In this method, scientists identify a gene that may affect a behavior, then interrupt or knock it out in a specially bred strain of mice. In this case, the effects of nicotine on the knockout mice and normal mice were compared, and the mice without the beta2 subunit lacked some responses to nicotine.

In work to identify receptor subunits that were sufficient to cause nicotine dependence, Lester's group examined the "partner" subunit, alpha4. But instead of experimenting with a knockout mouse, they developed "hypersensitive knock-in" mice. Lester's group replaced a naturally occurring bit of DNA with a mutation that changed a single amino acid in a single gene among the mouse's 30,000 genes. That change was enough to make the alpha4 subunit highly sensitive to nicotine.

Mouse Studies Lead the Way

Lester's group first selected the proper mutation with tests in frog eggs and cultures, then bred the strain of mice. As they hypothesized, the mice with the re-engineered alpha4 receptor proved to be highly sensitive even to very small doses of nicotine that didn't activate other nicotinic receptor types. This finding shows the alpha4 subunit is a prime candidate to be studied at the molecular and cellular level, says Lester, "and it may be a possible target for developing a medication that would reduce the release of dopamine caused by nicotine, and hopefully reduce nicotine's addictive grip."

Knockout mice leave nothing to measure, explains Lester. Knock-in mice have the advantage, he says, of isolating and amplifying the "downstream" chain of molecular signals within nerve cells that occur after nicotine activates its receptors. These signals, when repeatedly stimulated by nicotine, eventually cause the-as-yet unknown, long-term changes in nerve cells that are presumably the biological basis of addiction.

Goal: Therapy Against Addiction

Lester and his colleagues are now tracking down those signals, molecules, and newly activated genes, using their hypersensitive mice. The eventual hope: one of those signals might provide a molecular target that can be specifically blocked, providing a therapy against addiction. This strategy would resemble modern cancer drugs, such as Gleevec, which block only specific signaling molecules needed by proliferating cancer cells. Hypersensitive knock-in mice may also prove useful in gaining further insight into diseases such as epilepsy and Parkinson's disease.

Lester is optimistic about ultimately defeating nicotine's addictive power. "It's a complicated pathway that still must be broken down into individual steps before we can understand it fully," he says, "but I personally believe that nicotine addiction will be among the first addictions to be solved, because we already have so many tools to study it."

This research was supported by the California Tobacco-Related Disease Research Program, the W. M. Keck Foundation, the Plum Foundation, the National Institute of Mental Health, the National Institute of Neurological Disorders and Stroke, and the National Institute on Drug Abuse.