February 25, 2005

‘Speed’ can make protein run backward

Featured Image

Aurelio Galli, Ph.D., left, and Kristopher Kahlig, Ph.D., were the lead authors on the paper exploring how amphetamine makes neurons release large amounts of dopamine.
photo by Dana Johnson

‘Speed’ can make protein run backward

In the current online edition of the Proceedings of the National Academy of Sciences, researchers from Vanderbilt and Columbia University report that amphetamine, or 'speed,' makes neurons spew out dopamine by a previously unknown mechanism.

The team, led by Aurelio Galli, Ph.D., assistant professor of Molecular Biology and Biophysics, found that amphetamine causes dopamine to rush out of the cell through a channel, or pore, in the dopamine transporter (DAT) — a protein that normally sucks dopamine up from the extracellular space and transports it into the cell. This unexpected reversal of DAT function accounts for how amphetamine causes massive dopamine release and may offer an explanation for the psychoactive effects of amphetamine.

The results also suggest a potential therapeutic target for treating amphetamine addiction and disorders involving altered dopaminergic function (i.e., attention-deficit disorder and schizophrenia).

Under normal conditions, dopamine neurons release copious amounts of dopamine in tiny packets, called vesicles. A single vesicle can spit out around 10,000 molecules of dopamine. The DAT is responsible for sucking up excess dopamine from the extracellular space, so that it can be recycled inside the cell for later use.

Previous research has shown that when amphetamine is present, dopamine can escape through the DAT in a slow trickle: the DAT brings in a molecule of amphetamine and shuttles a molecule of dopamine out. However, this slow exchange mechanism of the DAT cannot account for the massive amounts of dopamine released in response to amphetamine.

In addition to the plodding transporter activity, the DAT also has a faster channel-like activity, but it has remained unclear what causes this channel to open.

Positing that amphetamine may affect the channel-like activity of the DAT, Kristopher Kahlig, Ph.D., a postdoctoral fellow at Vanderbilt and first author on the study, performed the technically demanding experiments to measure amphetamine-induced dopamine efflux (outward movement) in cells that carried the human version of the DAT and in cultured mouse dopamine neurons.

By coupling the standard patch clamp technique, which measures electrical currents in patches of neuronal membrane, with microamperometry, a carbon fiber that can detect the oxidation of dopamine molecules, Galli and colleagues counted the number of dopamine molecules that crossed the cellular membrane through a single transporter.

“The first thing we noticed was that dopamine was absolutely different from amphetamine,” Galli said.

When dopamine was present on both sides of membrane, there was no channel activity. However, when amphetamine was present on one side and dopamine on the other, the channel opened and large, brief 'shots' of dopamine were released.

These millisecond 'shots' of dopamine appeared similar in magnitude to the release of dopamine from a single vesicle, thus explaining the large increases in extracellular dopamine following amphetamine exposure.

“Dopamine is not able to stimulate the channel,” Galli said. “So we don't think that the channel-like activity participates in the physiological behavior of the DAT, but it can explain the psychoactive properties of amphetamine.”

Because amphetamine and dopamine have different effects on the DAT, this may allow researchers to treat addiction by shutting down the drug-related channel activity while leaving the normal, physiological transporter activity alone.

For a long time, the DAT has suffered an identity crisis: Is it a transporter? Is it a channel? This new study suggests that both may be true.

“For the first time, we show that a transporter can be both,” Galli said. “But we didn't define 'what' is making the transporter behave as a channel.”

To figure that out, Galli is currently investigating whether amphetamine causes chemical changes to cellular proteins that affect the opening of the channel.

The long-term goal, said Galli, would be to “try to find compounds that block one (activity) but not the other one, so that we can find a therapy for addiction.”

Kristopher Kahlig, Ph.D., is the first author on the PNAS paper. Other co-authors on the paper were: Francesca Binda, Ph.D., Habibeh Khoshbouei, Ph.D., Randy D. Blakely, Ph.D., and Douglas McMahon, Ph.D. of Vanderbilt, and Jonathan A. Javitch. M.D., Ph.D., of Columbia University. The research was supported by the National Institutes of Health and the Center for Molecular Neuroscience.