UNIVERSITY OF TEXAS SOUTHWESTERN MEDICAL CENTER AT DALLAS
The broad, long-term objectives of this project are to gain novel knowledge about a highly specialized calcium (Ca2+) channel - the inositol (1,4,5)-trisphosphate receptor (InsP3R). The proposed studies are fundamental to understanding Ca2+ signaling and may provide information relevant to multiple human diseases. My laboratory established functional expression of rat InsP3R1 in Sf9 cells and characterized properties of wild type and several mutant forms of recombinant InsP3Rl. We will continue to take advantage of the unique opportunity to measure functional activity of recombinant InsP3R and extend our structure-function analysis to other InsP3R isoforms. The specific aims of the proposal are: 1. To compare functional properties of different InsP3R isoforms in identical experimental conditions. The rat InsP3R1, InsP3R2, InsP3R3, D. melanogaster, and C. elegans InsP3R isoforms will be expressed in Sf9 cells and characterized in planar lipid bilayers. 2. To identify structural determinants responsible for the InsP3R1 modulation by Ca2+. Ca2+-binding and functional experiments will be performed to search for novel InsP3R1 Ca2+ sensor mutants. Ca2+ modulation of InsP3R-1/3 and lnsP3R-3/1 chimeric proteins will be analyzed in planar lipid bilayers to identify the locations of Ca2+-modulatory sites in the InsP3R sequence. 3. To characterize InsP3R modulation by PKA phosphorylation. Effects of PKA on InsP3R1 SII(+/-) isoforms, InsP3R2, and InsP3R3 channels will be evaluated. The PKA phosphorylation sites in InsP3Rsequence will be mutated and the sensitivity of generated mutants to PKA modulation will be analyzed. InsP3R1-PKA association will be investigated in biochemical experiments. 4. To determine an importance of InsP3R1 modulation by PKA in vivo. Dopamine-induced responses in striatal medium spiny neurons will be studied by Ca2+ imaging. Experiments with striatal neurons from DARPP-32 and D2 receptor knockout mice will be performed.