An elevation of the free calcium concentration in the cytoplasmic compartment is an integral component of the mechanism by which cells respond to hormones, growth-factors and certain neurotransmitters. D-myo-lnositol 1,4,5-trisphosphate (IP3) is an intracellular messenger mediating the hormonal mobilization of Ca2+ from intracellular stores. This molecule interacts with a specific receptor (IP3R) that has been purified and shown to be a ligand-gated Ca2+ channel. The central theme of this proposal is to study the structure, function and regulation of IP3 receptors. A major hypothesis to be tested is that interactions between the C- and N-terminal domains are fundamental to the mechanism by which ligand-binding leads to channel opening.
The specific aims of the proposal are to investigate: 1] The role of the C- terminal domain in channel gating. Deletion of 60aa from the C-terminal tail or 10aa from the cytosol- exposed loop between TM domains 4 &5 cause loss of channel function. Point mutants will be made to locate the critical amino acids involved in both regions. GST-fusion proteins and targeted cysteine cross linking studies will be used to test the hypothesis that regions of the C-terminus, TM4.5 loop and N-terminal domains are in close association. 2] The role of the N-terminal suppressor domain in channel gating. Deletion of aa1-224 of the N-terminus results in a marked stimulation of IP3 binding but loss of channel function. Mutagenesis will be used to identify critical amino-acids in this region. Conformational changes induced by IP3 in the ligand-binding domain will be studied using intrinsic tryptophan fluorescence and FRET methods. 3] Identify residues between transmembrane domains 5&6 which play a key role in channel function. Site-directed mutations will be made to identify residues that are part of the vestibule of the pore or the pore itself. Conductance properties and ion selectivity of the mutants will be investigated using electrophysiological approaches utilizing patch-clamped nuclei. These studies are intended to provide insights into the molecular architecture of the conduction pore. 4] Identify highly reactive thiol groups in the IP3R. Cysteine substitution and gel-shift assays using large maleimide polyethylene glycol derivatives will be used to identify surface accessible thiols in the IP3R. Changes in accessibility will be used as a probe to monitor conformational changes in the IP3R in native membranes. The long-term goal of this proposal is to understand how IP3R channels function at a molecular level and to use this knowledge to understand the mechanism by which cells generate the complex spatial and temporal patterns in Ca2+ signaling that underlie a multitude of physiological processes as diverse as cell division, cell proliferation, apoptosis, fertilization, development, secretion, smooth muscle contraction, memory and learning.
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