Inositol 1,4,5-triphosphate (InsP3) is a ubiquitous second messenger generated through the hydrolysis of phosphatidyl inositol 4,5 bisphosphate by phospholipase C. InsP3 binds to a family of very similar receptor proteins localized to the endoplasmic reticulum and mediates the release of intracellular calcium through an intrinsic calcium channel. Intracellular calcium release via InsP3 has been implicated in a multitude of cell biological processes ranging from mesoderm induction in the developing xenopus to increased contractile force in cardiac muscle. Three highly similar isoforms of the receptor family have been cloned and sequenced. Mutagenesis studies have enabled the assignment of a three domain model for the receptor's functional organization consisting of a NH2-terminal ligand binding domain, a C-terminal calcium channel domain and a central coupling or regulatory domain. The coupling domain is the least conserved region among the receptor types and encompasses sequences thought to be involved in the regulation of channel function such as alternate splicing, phosphorylation, nucleotide and calcium binding. Despite the overall high degree of structural similarity, the individual receptor species exhibit significant functional heterogeneity in that they have greatly differing affinities for InsP3. Numerous studies have demonstrated that there is a large variability in the sensitivity of different preparations to InsP3 suggesting that variations in ligand sensitivity may result from the structural heterogeneity of the receptor family members. In this proposal, the function of specific single InsP3R isoforms will be defined by incorporating native and recombinant channel proteins into artificial planar lipid bilayers.
The specific aims of this proposal are to test: 1) That specific isoforms of the InsP3R can be isolated into homogenous populations; 2) Determine if different homologues of the native receptor channel are functionally heterogenous; 3) Test the hypothesis that single recombinant InsP3 receptor calcium channels are functionally identical to their natively expressed counterparts; 4) To test the hypothesis that sequence heterogeneity within the three principle domains of the InsP3R subtypes confers isoform-specific functional differences.
