The vertebrate olfactory system is remarkable for its ability to detect and discriminate between thousands of small organic molecules commonly known as odors. The molecular and physiological mechanisms employed by the primary sensory neuron to accomplish this recognition task shares many features with other cellular signal transduction systems in the nervous system. The olfactory receptor neuron provides a superior model cell for examining these general issues of cell regulation and signalling. This proposal seeks to investigate three main issues to achieve a quantitative understanding of these molecular strategies. Experiments will utilize a newly developed stimulus delivery system that enables the investigator to completely control the time course and intensity of olfactory stimuli impinging on a single cell, while the electrical responses of that cell are monitored. With this level of control it is anticipated that the first quantitative measurements of the coupling reactions in the signal transduction cascade can be obtained. Further, a thorough characterization of the phenomenon of adaptation will be developed, again using closely controlled stimuli. These experiments are expected to result in new insights into the detailed workings of G-protein coupled signalling systems, including those found in the retina, at synapses and in hormonal effectors. Since so many cell regulatory systems operate through these G-protein coupled cascades this data will have wide application. The qualification of adaptation may help to understand such clinically crucial issues as drug tolerance and drug efficacy. An additional aim of this proposal is to understand how the individual olfactory neuron is capable of responding to several odors. Does this neuron express one, promiscuous receptor or multiple specific receptors. The answer to this straightforward question is fundamental to developing models for stimulus mapping in the brain as well as for appreciating the initial development and continuous regeneration of the olfactory system. A new technique, single cell PCR, will be utilized to investigate this crucial issue. The results could have important implications for understanding phenotype determination at the cellular level and for delineating the relationship between the peripheral epithelium and the central structures of the olfactory system. The results will also provide the fist structure-function data for at least some members of the enormous olfactory receptor gene family of receptors. Because olfactory receptors are able to make fine discriminations between molecules with very similar structures there is the opportunity to understand how a single amino acid change in the receptor protein can alter binding affinities for various ligands. This may have important ramifications for rational design of drugs targeted at receptors of known structure.
