Stuart Firestein - US grants

A commonly observed phenomenon in biological systems is the waning of response amplitude when the stimulus signal is continuously present. This phenomenon was first observed in sensory systems where it was called adaptation, and later was noted to be a common feature of drug, neurotransmitter and hormone receptors where it is known as desensitization. Adaptation and desensitization are similar effects, one occurring at the systems level and one at the cell-molecular level of organization. It now seems probable that common mechanisms may have developed in both sensory and other receptor systems for mediating this effect. The olfactory receptor neuron is a unique model system for understanding both effects beacuse it respnds to envirnmental stimuli (sensory function) and these stimuli are chemical in nature (pharmacological ligand-receptor frunction). Recently we have shown that olfactory transduction is mediated by a second messenger system similar to those operating in phototransduction and in - adrenergic receptor mediated signal transduction. In analogy with these systems, one of the several steps in this enzyme cascade may be the molecular site of an adaptation feed-back link in olfactory receptor neurons. The entrance of Calcium ions during the odor response also suggests a possible role for Ca in producing desensitizations. The methods to be employed include whole cell patch clamp recordings of the ion currents generated by exposure to odors in single receptor neurons. The effect on these currents of various pharmacological events which interact with component enzymes in the second messenger cascade will indicate the likely site(s) and mechanism(s) underlying adaptation. Additionally single channel recording methods will be used to measure the activity of cAMP gated channels under conditions which are hypothesized to activate and inactivate this channel. Mechanisms by which signal transducing systems are modulated is a question of general relevance in cell biology and is of unique interest in the olfactory system where adaptation plays an important physiological and behavioral role for the organism. At the molecular level desensitization is an important factor limiting the clinical efficacy of drugs delivered for long periods. If, as we postulate. common mechanisms underlie this widespread biological phenomenon, then a mechanistic appreciation is essential to devising interventions which either increase or decrease response adaptation.

9312177 Firestein This two-year award will support U.S.-Italy cooperative research between Stuart Firestein of Columbia University, and Anna Menini of the University of Genoa in Italy. They will collaborate on stimulus-response relations in olfactory neurons. Signal transduction systems are often measured by dose-response relationships. This critical quantitative measure is unavailable for olfactory receptors, due largely to the difficulty of accurately delivering a stimulus of known concentration. This project seeks to provide this data by applying a novel means of stimulus delivery, in which a single living cell can be completely immersed in a particular odor solution while its electrical response is monitored. From this data, it will be possible to construct the dose-response relations of individual olfactory cells to a variety of odor stimuli. Both investigators are established and highly regarded, the problem is fundamental to sensory neuroscience, and the collaboration exploits complementary skills with a novel and unique approach. The collaboration has the potential to have a major impact on understanding chemosensory functions. * * *

This project will make use of a new method for functionally expressing members of the odor receptor gene family. An adenovirus vector will be utilized to drive over expression of cloned receptors in mice, where they can be assayed for their specific odor affinities. In this way the "molecular receptive field" of these receptors will be defined for a set of odorant ligands. Receptors with related gene sequences will be screened to determine if they also recognize related or overlapping sets of odor ligands. Because the cells expressing the cloned receptors also express GFP it is possible to record from identified single cells dissociated from the olfactory epithelium. With single cells stimuli of carefully controlled concentration and duration can be delivered, allowing for the development of dose response relations for each of the ligands that a receptor may be able to bind. It will further aid in the identification of potential antagonists and other molecular competitors that may act at these receptors. Antagonists are an important tool in the studying structure function relations between receptor and ligand. The overall aim of the project is to understand in some detail how the thousand or so odor receptors manage the task for detecting and discriminating some 10,000 odor ligands. By focusing on a subset of these receptors in the mouse, where it is possible to correlate these data with information from genetically targeted animals, it is expected that some general principles will emerge, both at the molecular level of the odor receptor protein, and at the systems level of olfactory coding. Odor receptors comprise the largest family of G-protein couple receptors in the mammalian genome. As such they represent a kind of "natural" experiment in which variations in receptor structure due to differences in amino acid sequence can be related to alterations in receptor-ligand affinity. Because the odor receptors are in the same genetic superfamily of receptors as those for neurotransmitters, hormones, drugs and many peptides, understanding the relationship between structure and function in these receptors could lead to important new principles for rational drug design and for receptor modifications. Many neurological diseases are the result of deficient receptors for neurotransmitters or neurohormones. These results will speak directly to those causes.

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