Through the turnstile

Transporter proteins mediate initial actions of psychostimulants

In the moments that follow a hit of cocaine, drug molecules pulse through the body, landing ultimately in the grooves of their molecular targets -- neurotransmitter transporters.

The transporters are something like clanking turnstiles, says Randy D. Blakely, Ph.D. Dopamine, serotonin, and other chemical messengers (neurotransmitters) pass through them, moving out of the void between two nerve cells -- the synapse -- and back inside the "presynaptic" neuron, where they can be re-packaged for future use.

Cocaine blocks the turnstile specific for dopamine, leaving dopamine hanging out in the synapse, where it can continue to signal. This overload of dopamine in the brain's "reward system" is key to the pleasurable and addicting effects of cocaine, Blakely says.

Amphetamines, too, affect the dopamine transporter's activity, but instead of blocking the turnstile, they seem to send it spinning in reverse, spewing dopamine out into the synapse.

"The dopamine transporter (DAT) has historically been the number one target thought by many to underlie at least the initial actions of amphetamines and cocaine, in terms of their psychostimulant and rewarding properties, and in terms of their addictive liability," Blakely says.

Blakely and fellow Vanderbilt investigators Louis J. DeFelice, Ph.D., and Aurelio Galli, Ph.D., make a formidable team in the effort to understand the transporter targets for cocaine, amphetamines, and other psychostimulants. They've made inroads into characterizing how these transporters work at the molecular level and how genetic changes in these proteins may contribute to disease.

DeFelice and colleagues discovered a few years ago that in addition to moving neurotransmitter molecules across the membrane, the DAT also alters the electrical properties of the neuron, affecting its ability to send signals. The challenge now, he says, is to figure out how a transporter can have such a split personality: how does the cell regulate whether it has transporter-like or electrical channel-like properties?

Galli and collaborators at Vanderbilt and Columbia University have been pursuing the question of how amphetamines affect the DAT. Several years of studies have revealed that amphetamines promote the redistribution of the DAT off the cell surface, that they cause dopamine to rush out of the cell through a channel in the transporter, and that a modification of the transporter called phosphorylation is likely required for amphetamine actions.

Galli's findings suggest that the DAT can be both transporter and channel. And because amphetamine and dopamine have different effects on the DAT, it may be possible to treat addiction by shutting down the drug-related channel activity while leaving the normal, physiological transporter activity alone, Galli says.

"We think we've identified a region of the transporter molecule that is of real importance for amphetamine action," he adds. "It might be a good target for new therapeutic agents that block the effects of amphetamine-like psychostimulants."

Because proteins like the DAT play a role in the initial signaling for drugs of abuse, they may participate in predisposition for abuse liability, Blakely notes. "The key here is not to focus on the transporter but to focus on the system," he says. "There are a lot of molecules that control how dopamine works -- how it's made, how it's transported, how it's broken down."

To this end, Blakely's team has turned to a simpler model system, the nematode worm C. elegans. The investigators have engineered worms to express a fluorescent DAT, so that it can be visualized -- the worm's eight dopamine neurons "glow" green -- and followed in a living organism.

"We've been able to watch where the DAT goes in an intact nervous system, and how genetic mutations affect its localization," Blakely says. His team is continuing to use genetic approaches to screen for genes that regulate the DAT and that are required for supporting amphetamine-induced behaviors in the worm.

The transporter field was excited last year by the publication of the X-ray crystal structure for a bacterial transporter protein.

"This is the 'Rosetta stone' for our protein," Blakely says. "We are now taking our human dopamine and serotonin transporter proteins and threading their structures onto that backbone."

The serotonin transporter is a target for both cocaine and the amphetamine-like molecule MDMA -- Ecstasy. Having a structural model for the serotonin transporter is allowing the investigators to move very quickly to questions about where MDMA binds to the transporter, how that binding is different from antidepressants and from serotonin, and how it triggers the transporter to run in reverse, Blakely says.

"I think we're getting a handle on the molecular actions of these substances."

Blakely is the Allan D. Bass Professor of Pharmacology, professor of Psychiatry, director of the Center for Molecular Neuroscience, and an investigator of the Vanderbilt Kennedy Center for Research on Human Development. DeFelice is professor of Pharmacology. Galli is associate professor of Molecular Physiology & Biophysics.
- Leigh MacMillan and Melissa Marino