This is an application for renewal of funding study chemical sensing in Paramecium. This organism is amenable to multiple techniques for dissecting chemosensory transduction mechanisms in chemoreceptor cells: biochemistry, molecular biology, transmission genetics, electrophysiology, behavioral analysis of populations and individual cells. After we disrupt the cell and study its transduction components in vitro, we can often return our study to the level of the intact cell and ask whether our findings have relevance for the physiology of the cell. Also, we know the identity of stimuli, which bind to distinct receptors. In the past funding period, we have accomplished most and gone beyond some of our original specific aims which were to apply molecular, immunological, biochemical and fluorescent dye techniques to the study of the cAMP receptor and the plasma membrane calcium pump, which appears to participate in transduction pathways. We have partial clones of the cAMP receptor gene, and the full length gene for the plasma membrane calcium pump. We have subcloned and studied the pump's calmodulin binding domain. Highlights include There are not 1 but at least 3 transduction pathways, 2 of which involve the calcium pump; attractant stimulus NH4CI has no receptor and rapidly alkalinizes the cell; while all attractant stimuli hyperpolarize the cell, only glutamate increases cAMPI and very rapidly; some chemoreceptors probably are GPI anchored and not integral membrane proteins; biotin is an attractant and its receptor likely is GPI anchored cloned; attractants hyperpolarize cells and the conductances initiated by biotin and acetate have been partially characterized. There is a sustained outward conductance (possibly the pump current) and a large inward conductance for the off-response in biotin; there is an initial inward calcium and outward K conductance in acetate with a small inward conductance for the acetate off-response. The calcium pump appears to sustain the hyperpolarization in both biotin and acetate. We used mutants with conductance defects to test these ideas. We propose to test three hypotheses that arise from our studies. 1) Chemoreceptors for biotin and glutamate are GPI anchored proteins. 2) Chemosensory transduction in 2 pathways in modulates a calcium pump to generate a hyperpolarizing conductance. Gene structure suggests that these modulations involve PKA, PKC or calmodulin. 3) Biotin and acetate induce distinct sets of Conductances, which we will test using voltage clamp analysis of normal, mutant and transformed cells. We will use electrophysiological, biochemical, molecular biology, and immuno-techniques in a collaborative effort between two laboratories. These studies should inform the research of others who study sensory transduction in olfaction, taste, chemotaxis and other signaling stems.
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