The human brain-and how it works-has intrigued researchers for centuries. Recent advances in biotechnology have opened new worlds of research opportunities, leading to critical breakthroughs in our understanding of human brain function. At the University of Connecticut Health Center, scientists are focusing on one of two major classes of neurotransmitters that govern brain activity: the GABAA receptors.
Communication among neurons in the brain is mediated by chemical messenger molecules called neurotransmitters, which are released from one cell to act on another cell. This process is called neurotransmission. Once the neurotransmitter molecule reaches the target cell, it binds to its receptor, and its influence on that cell is mediated by the specific type of receptor found on the cell surface. Each neurotransmitter will act only through its own class of receptor, but within a given class of receptor there may be several subclasses, each of which can have slightly different effects.
There are two major classes of neurotransmitter receptors, excitatory and inhibitory, and activity within the brain can be viewed as a fine balance between these processes.
"Gamma-aminobutyric acid (GABA) is one of the most common and potent inhibitory neurotransmitters," says Hermes Yeh, professor of pharmacology and neurology at the University of Connecticut Health Center. "I'm interested in the GABA receptors because of their potential to be modulated, their involvement in disease processes, and the potential for therapeutics that act on GABA receptors."
Once GABA is released from a cell, it can potentially influence three subtypes of receptor: GABAA, GABAB, and GABAC. GABAA, the receptor in which Yeh is most interested, mediates the fast, moment-to-moment synaptic transmission that is most often associated with brain activity.
"GABAA is important," explains Yeh, "because studies performed more than 20 years ago, as well as ongoing studies, have shown that it is the target of many drugs, such as those that are used to treat epilepsy and anxiety." These drugs include the anticonvulsants, benzodiazepines (e.g., valium), and general anesthetics (e.g., barbiturates).
Alcohol also modulates the GABAA receptor. "At high doses, alcohol has effects similar to those of many anesthetics," notes says Yeh. "At low concentrations, alcohol has a sedative effect."
These drugs generally act on neurons to decrease the cell's excitability, by enhancing the action of the GABAA receptor. This receptor is associated with an ion channel that allows chloride molecules into the cell. "The prevailing thought is that alcohol and many general anesthetics exert their effects directly or indirectly by influencing the flow of chloride ions through the channel," explains Yeh. "If the channel is kept open longer or allowed to open more often, chloride levels inside the neuron will increase, the neuronal membrane will be hyperpolarized, and the ability of that neuron to be excited will be decreased."
Alcohol, valium, or general anesthetics may not affect all parts of the brain equally, notes Yeh. "Certain parts of the brain are more sensitive to the effects of these drugs than are others," he explains. "Is this because there are more receptors in those areas, or are there subtle differences in the receptors in different parts of the brain? These are questions that we've been working on. What we've found is that there are, indeed, different types of GABAA receptors, which have different sensitivities to drugs."
GABAA receptor is a complex assembly of proteins, made up of five protein subunits with a hole in middle-like a tube made of Legos-to form the chloride channel. The receptor spans the membrane of the nerve cell, so that the channel extends from outside to inside the cell and chloride can move in if the channel is open. There is a repertoire of at least 18 different subunits, all encoded by different genes, from which the GABAA receptor can choose its five.
The receptor subunits have been divided into six categories based on the molecular biology of their gene sequences. So far, six alpha subunits, four betas, three gammas, one delta, one epsilon, and three rho subunits have been identified.
"If the subunits joined together randomly, there would be thousands of possible combinations," notes Yeh. "It doesn't seem to work that way, however." Rather than just randomly combining any five of the subunits, there appears to be an order to the way the final receptor is formed. "Most of the GABAA receptors found in humans are made of two alphas, two betas, and one gamma subunit," he says. "Several of the other subunits are not very prevalent, but are nonetheless important. For example, the delta subunit is very uncommon, the alpha-6 subunit is found only in one type of cell (the cerebellar granular cell), and the rho subunits are found only in cells of the retina at the back of the eye and a few other brain regions, such as the cerebellum."
"One reason that different areas of the brain respond differently to drugs is that the receptors found on those cells differ," he explains. "Another reason is that the receptors are differentially distributed, that is, they are more prevalent in some areas of the brain than in others."
According to Yeh, researchers have used recombinant DNA techniques to discover that GABAA receptors made artificially of different combinations of subunits have different properties. "The work that we do in my laboratory focusses on real cells rather than artificial constructs," he says. "Studying real cells, we have found that they actually express GABAA receptors made up of different subunits that can have different pharmacological profiles-they respond differently to different drugs."
For example, the alpha subunit is necessary for benzodiazepines to work, and different alpha subunits can confer different sensitivities to drugs of this class. "This motivated me to study neurons in different parts of the brain, to examine how different their responses are, and how that relates to differential expression of GABAA receptors," Yeh explains. "We're interested in revealing the molecular basis for differences in drug sensitivity and other functional properties of native GABAA receptors. We are also trying to determine if neurons that express the genes encoding the particular subunits actually produce the proteins (i.e., make the corresponding mRNAs) and assemble them into functional GABAA receptors."
The challenge for Yeh's research group is to come up with a way to simultaneously study function of the receptor, gene expression, and protein production. "We can record electrophysiologically from cells to determine their function, and can study gene expression and protein production in tissues," he explains. "But brain tissue is heterogeneous, there are lots of different kinds of cells. To get to the level of detail we need, we must do all of this work on single cells. That's not easy."
His group has developed new techniques, and refined existing ones, to come up with a reliable method of obtaining the information they need from single cells. "We call it 'combined patch-clamp recording and single-cell mRNA expression profiling'," he explains.
"The first step is to do a patch-clamp recording of a single cell to get the physiological and pharmacological profile of the cell," he explains. Patch-clamp recording is a technique by which a tiny electrode is attached to a single cell, allowing the investigator to record the electrical signals produced by the cell. "Then we aspirate the cell into the recording pipet (similar to a tiny medicine dropper) and transfer it to a test tube for a procedure called 'reverse transcription,' which allows us to work with a purified sample of the genetic material (RNA) from the cell." After researchers have purified the RNA, they use another molecular biological technique, called PCR (polymerase chain reaction) to amplify it one-million-fold or more.
"Then we have the equivalent of the genetic material from more than one million cells," continues Yeh, "except that it's all from one cell, the same cell that we did the patch-clamp recording on." Using molecular biology techniques, his group can pick out from a single cell the RNAs that encode the GABAA receptor subunits. "In this way we can get molecular biological and functional data from the same single cell," he says.
"To kick it up one more notch, we've been developing and refining the procedure of profiling protein expression for about one year, and we have some encouraging results," he continues. "We hope that soon we will achieve our goal-to study function, gene expression, and protein expression all in a single cell."
One practical application of Yeh's work is to determine the relationship between the different subunits of receptors and the sensitivity of those receptors to drugs. "If we could develop drugs that are specific for receptors comprising certain subunits, those drugs would be more potent and produce fewer side effects," he explains. "And our technique can be used with a variety of cells, not just neurons, so it has possible applications in virtually every field of biomedical research."-- Lisa Christenson, science writer
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