November 19, 2004

DAT’s incredible: Cellular vacuum cleaner has new role

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DAT’s incredible: Cellular vacuum cleaner has new role

After a neurotransmitter has completed its job of sending a chemical message between two neurons, it is tossed, like a cigarette butt from a car window, out into the void between the cells (the synapse). Luckily, the neuron that sent the chemical message (the presynaptic neuron) is equipped with special molecular “vacuum cleaners” called transporters that can clean up the resultant mess.

“Until very recently, transporters were thought of as 'benign vacuum cleaners,'” said Louis DeFelice, Ph.D., professor or Pharmacology. “They just vacuumed up the transmitter after it was released, put it back inside, and that was their job.”

However, a new study by DeFelice and colleagues reveals that there's more to a transporter's function than cellular housekeeping.

In the study, DeFelice and colleagues provide the first direct evidence of a long-suspected, but never before measured, activity of the dopamine transporter (DAT) in neurons isolated from the microscopic worm, C. elegans. They found that transporters actually influence the electrical properties of the neuron, affecting its ability to fire. These results appeared in the Nov. 1 online edition of the Proceedings of the National Academy of Sciences.

“Suppose you plugged in the vacuum cleaner and started vacuuming, but the lights in the room got brighter,” DeFelice explained. “You would realize that you weren't only vacuuming up the (debris), but you were affecting the electrical properties of the house.”

“That's exactly what we discovered … that (the DAT) not only transports dopamine back into the presynaptic terminal from whence it came, but it also affects the electrical properties of the synapse. In other words, it generates a current.”

An electrical current in a neuron results from large amounts of charged molecules (sodium and chloride, for example, as well as dopamine itself) rushing into and out of a cell. This is usually accomplished by the opening of a channel. When open, a channel acts like a floodgate, allowing a deluge of charged ions or molecules to pass through.

Transporters, on the other hand, are sluggish machines, akin to revolving doors. Typically, transporters can only transport a few molecules at a time — not enough, scientists thought, to create an electrical current of any size.

But researchers had seen hints of this activity associated with DATs in artificial systems — non-neuronal cells that are genetically engineered to express DAT protein — and in native cells — neurons that normally contain DAT. However, neither of these systems provided direct evidence of channel-like activity for the DAT.

C elegans, with its eight dopamine neurons, provided a simple, well-characterized model system in which to study DAT activity. And tools developed by Vanderbilt colleagues Kevin Strange, Ph.D, and Michael Christensen (culture techniques for C. elegans embryos) and Richard Nass, Ph.D., (a method to visualize dopamine neurons using green fluorescent protein), made it possible to use the worm to explore DAT's elusive channel-like properties.

Lucia Carvelli, Ph.D., a postdoctoral fellow in DeFelice's lab and first author on the paper, isolated individual dopamine neurons from the worm, cultured them and analyzed channel activity using a standard patch clamp technique.

Carvelli and DeFelice observed single-channel events when dopamine was applied to the cells that contained DAT. No channel activity was detected in cells that lacked DAT. In addition, the uptake of dopamine by the DAT depolarized the neurons by about 10 mV, making them more liable to fire.

As the first study to show authentic single-channel activity in a neurotransmitter transporter, these results are likely to affect the way scientists think about transporters in the future.

The challenge now, said DeFelice, is to figure out how a transporter can have such a split personality; how does the cell regulate whether the DAT has transporter-like or channel-like properties?

“Now we have a model where we can ask this question at a molecular level. We can add or remove genetic components to see if they regulate it,” DeFelice said.

Since dopamine neurons are involved in normal and abnormal human behavior and disease (i.e., in the so-called “pleasure pathways” and Parkinson's disease), DeFelice's work unraveling the mysteries of the DAT may have broad implications for a plethora of human conditions. Also, DAT is a major site of drug action (for example, cocaine prevents dopamine uptake by blocking DAT, and amphetamines release dopamine via DAT), so understanding transporter function is key to understanding drug abuse.

In addition to the DAT, there are transporters for many other neurotransmitters (i.e., serotonin, norepinephrine, glutamate) and nutrients (i.e., zinc, iron, some sugars). Interestingly, many of these transporters appear structurally and mechanistically similar to DAT.

“Nature always uses the same tricks over and over,” said DeFelice. “So I'm certain that what we learn will spill over, not only to other neurotransmitter transporters, but to the entire world of transporters. It's a safe bet.”

Other Vanderbilt authors on the study were Paul W. McDonald, and Randy Blakely, Ph.D. The research was supported by grants from the National Institutes of Health.