Regulation of nicotinic signaling
Initial Award Abstract
Tobacco use is thought to be the leading cause of premature death in the US. The major addictive component of tobacco is nicotine, placing it at the center of this health tragedy. The immediate target of nicotine is a family of signaling proteins in the nervous system called nicotinic acetylcholine receptors. Activation of receptors produce a variety of consequences in brain cells because the receptors let calcium enter the cells and calcium controls many processes. The consequences of nicotinic stimulation, therefore, depend critically on where the receptors are and on the kinds of calcium-sensitive components tethered nearby. Recently it has been found that the receptors are associated with molecular scaffold that can tether specific components in the immediate vicinity. The backbone of the scaffold employs a protein specifically designed to bind other proteins. Preliminary evidence suggests that the scaffold not only organizes components near the receptors but also plays a major role in promoting functional nicotinic innervation of the cell. This enhancing effect does not appear to involve changes in the number of receptors themselves but rather appears to represent an ability of the scaffold in one cell to indirectly affects events in another cell through some kind of interaction. A candidate molecule that might serve this purpose is neuroligin. Preliminary results indicate that neuroligin is expressed in the cells and that it can cause a dramatic increase in nicotinic input to the neurons. It is not known, however, whether the neuroligin effect depends on the scaffold.
The experiments proposed here have three aims. First will be analysis of how the scaffold in neurons affects nicotinic input. This will be done by using both electrical recordings to monitor electrical signals and using fluorescence microscopy to visualize the contacts made between cells. The scaffolds will be disrupted by introducing appropriate components into the cells, and the effect will be determined of receptor function, electrical signaling to the cells. Candidate molecules mediating the effects will also be tested. Second, experiments will be carried out on cells manipulated to express different kinds of neuroligin so that we can determine how neuroligin boosts nicotinic signaling the cells. This will again include both electrical recording and fluorescence microscopy. Related questions will be the role, if any, of the scaffold in the neuroligin effect, possible interactions with components on nearby cells, and how the neuroligin comes to be specifically associated with the receptors themselves as suggested by preliminary results. The third aim entails similar experiments on a different class of cells in culture to determine how pervasive these mechanisms are for regulating the development and maintenance of nicotinic signaling and input.
The results obtained will provide new and important information about the molecular mechanisms that promote and sustain nicotinic signaling in the nervous system. They will shed new light on the cellular apparatus that determines the consequences of nicotinic stimulation. This is likely to have significant biomedical consequences because of the central role of nicotinic receptors in tobacco-related diseases. |