By David N. Leff
This coming Monday in Honolulu, Cornell University molecular biologist George Hess will report his latest discovery to the American Chemical Society¿s Pacifichem International Congress. His presentation is titled: ¿Illuminating reactions controlling communication between brain cells, using light-activatable neurotransmitter precursors (caged neurotransmitters).¿
He will tell the conference audience, ¿The reactions mediated by neurotransmitter receptors in the membrane of the 1012 [one trillion] neurons in the mammalian nervous system that control communications between these cells are basic to brain function.¿
Hess and his Cornell co-workers are exploring the mechanism of these reactions on the surface of neurons by means of biologically inactive precursors of neurotransmitters ¿ i.e., ¿caged¿ neurotransmitters. Their focus is on nicotinic acetylcholine and its receptor (AchR). These are among the central nervous system¿s top-gun proteins, carrying the brain¿s ceaseless stream of messages, from synapse to synapse, throughout those trillion cells. They form transmembrane ion channels.
One communication ¿virus¿ obstructing this AchR system is cocaine.
Hess and his team have pinpointed several RNA compounds that prevent cocaine from inhibiting AchR. They did so by deploying their novel ¿laser-pulse photolysis¿ technique, which, he explained, ¿lets us look at an ensemble of molecules ¿ not just a single channel or a single molecule ¿ during the split-second reactions that relay electrical signals through the nervous system.¿
He is senior author of a paper in the current Proceedings of the National Academy of Sciences (PNAS), dated Dec. 5, 2000. It¿s titled: ¿Mechanism-based discovery of ligands that counteract inhibition of the nicotinic acetylcholine receptor by cocaine and MK-801 [an experimental anticonvulsant called dizocilpine].¿
Laser-Pulse Photolysis ¿ User¿s Manual
Another of the PNAS paper¿s co-authors, research associate Susan Coombs, tells how their system works: ¿A protecting group is attached to a neurotransmitter ¿ the chemical signal. This produces a caged neurotransmitter,¿ a molecule that has no effect on the receptor proteins. Thus, it can be mixed with a cell without triggering reactions, and achieve equilibrium between the caged compound and the receptors on the surface of a single cell.
¿A single pulse of laser light,¿ Coombs continued, ¿cleaves the protecting group from the neurotransmitter within microseconds to milliseconds ¿ while these proteins are in the receptors¿ open¿ state. The active neurotransmitter then binds to the receptors, causing them to form open channels through which electrical currents flow. This,¿ she pointed out, ¿gives information about the balance between open and closed channels ¿ information about whether cocaine, the RNA ligands or hundreds of known therapeutic agents or abused drugs tip the balance toward the open or closed channel states of the receptors.¿
Coombs made the point that this method ¿increased the signal associated with the opening of receptor-formed transmembrane channels 1,000-fold over what was available previously. The technique,¿ she added, ¿made it possible for the first time to resolve the steps of the mechanism associated with the binding of neurotransmitters to the receptors, the opening of transmembrane channels, and the effect of activators and inhibitors on these steps.
¿In the PNAS paper,¿ she went on, ¿we report that on the basis of measurements using the recently developed techniques, we arrived at a mechanism for the inhibition of the AchR by cocaine. This membrane-bound receptor is a key protein in signal transmission between nerve cells. Upon binding a specific chemical signal ¿ acetylcholine ¿ secreted by an adjacent cell, the receptor opens a cation-specific [positively charged] transmembrane channel, and a signal is transmitted.
¿Our mechanism,¿ she observed, ¿indicated that cocaine binds with higher affinity to the closed channel than to the open-channel form, and thereby inhibits the receptor. If one can find compounds that bind better to the cocaine site on the open-channel form, they would not inhibit the receptor, and would prevent cocaine from binding. We have found such compounds,¿ Coombs said.
Post-doctoral physical chemist Amanda Gameiro, a PNAS co-author, recounted, ¿In this technique we have three components: the cells we are studying, which we lift from their dish and put in front of a U tube ¿ a device where we apply the caged neurotransmitter, a precursor of the neurotransmitter, which is inactive. It surrounds the cell, and then we have an optical fiber that photolyzes the compound, which binds and opens the channel. The time and space resolution,¿ she went on, ¿are very good because the caged neuron is already surrounding the cell; it¿s already there. This is the advantage of the laser pulse.¿
At Last: Mechanism-Based Drug Design
¿When we photolyze the caged, inactive precursor around the cell,¿ Gameiro continued, ¿it releases the neurotransmitter. And when we apply cocaine we can study how it affects these mechanisms, and determine its mode of action. Cocaine acts not just on the acetylcholine receptor,¿ she pointed out. ¿It acts on lots of other receptors and transporters. So basically, what cocaine does is in the case of the acetylcholine receptor, for example, it would bind to the receptor and hinder the opening of the channels, thus inhibiting it.¿
¿Now,¿ Hess observed, ¿mechanism-based drug designers can go to work developing treatments for cocaine poisoning [addiction], as well as neurological diseases such as epilepsy and Parkinson¿s.¿
At the Honolulu meeting, Hess will present examples of ¿elucidating the reaction mechanisms of the AcH receptors, for understanding the integrated response of neurons to excitatory and inhibitory neurotransmitters, for understanding the effect of therapeutic agents, abused drugs and disease-causing mutation of receptors on their function in mechanism-based drug design.¿
This effort, he suggested, ¿could proceed more rationally, knowing the exact roles and timing of all the chemical players at the junctions between neurons and muscle cells or between neurons and other neurons. Understanding of how neurons respond to the two kinds of neurotransmitters ¿ excitatory and inhibitory ¿ could be greatly expanded.¿